Identification, diagnosis, and treatment of neuropathologies, neurotoxicities, tumors, and brain and spinal cord injuries using electrodes with microvoltammetry

ABSTRACT

The present invention relates to devices and methods of use thereof for detection of biomolecules, in vitro, in vivo, or in situ. The invention relates to methods of diagnosing and/or treating a subject as having or being at risk of developing a disease or condition that is associated with abnormal levels of one or more biomolecules including, but not limited to, inter alia, epilepsy, diseases of the basal ganglia, athetoid, dystonic diseases, neoplasms, Parkinson&#39;s disease, brain injuries, spinal cord injuries, and cancer. The invention also provides methods of differentiating white matter from gray matter. In some embodiments, regions of the brain to be resected or targeted for pharmaceutical therapy are identified using sensors. The invention further provides methods of measuring the neurotoxicity of a material by comparing microvoltammograms of a neural tissue in the presence and absence of the material using the inventive sensors.

This application is a continuation-in-part application under 35 U.S.C.§120 of U.S. patent application Ser. No. 10/118,571, filed Apr. 8, 2002,now allowed, entitled, “Identification, Diagnosis, And Treatment OfNeuropathologies, Neurotoxicities, Tumors, And Brain And Spinal CordInjuries Using Microelectrodes With Microvoltammetry,” which claims thebenefit of U.S. provisional application Ser. Nos. 60/326,407, filed onOct. 1, 2001; 60/297,276, filed on Jun. 11, 2001; and 60/282,004, filedon Apr. 6, 2001, all now expired, all of which are herein incorporatedby reference.

FIELD OF INVENTION

This invention relates to the identification, diagnosis, and treatmentof neuropathologies, neurotoxicities, tumors, and brain and spinal cordinjuries using electrodes with microvoltammetry.

BACKGROUND OF THE INVENTION

The present invention relates to devices and methods of use thereof fordetermining the presence and concentration of chemicals in a cell,tissue, organ or organism. The invention relates to, inter alia,semiderivative voltammetric measurements and chronoamperometricmeasurements of chemicals, e.g. neurotransmitters, precursors andmetabolites, to identify, diagnose, and/or treat neuropathologies,neurotoxicities, tumors, and brain and spinal cord injuries.

Microvoltammetric indicator microelectrodes pass small but measurablecurrents while neurotransmitters and metabolites close to themicroelectrode surface undergo oxidation and/or reduction (Adams R N etal., 1982, Handbook of Psychopharmacology, pp. 1-74). When an electrodeis placed in contact with a solution a phase boundary is created thatseparates identical solutes into two different types. They are (a)molecules that are at a distance from the microelectrode and (b) thosemolecules that are close enough to participate in mutual interactionsbetween the surface of the microelectrode and the sample solutioninterface (Kissinger P T et al., 1996, Laboratory Techniques inElectroanalytical Chemistry, pp. 11-50). Collectively, theseinteractions are called electrochemistry.

Detection of electrochemical signals from solutions and from anatomicbrain sites is termed “faradaic” because the amount of the oxidativeand/or reductive species detected at the surface of the microelectrodemay be calculated by a derivation of Faraday's Law, the CottrellEquation, 1i_(t)=nFAC₀D₀ ^(1/2)/3.14^(1/2)t^(1/2), where i is current attime t; n is the number of electrons (eq/mol); F is Faraday's constant(96,486 C/eq); A is electrode area (cm²); C is concentration of oxygen(mol/cm³), and D is the diffusion coefficient of oxygen (cm²/s). Theproportionality between charge and mass of an electrochemical reactiondescribes the relationship between the charge of each neurochemical inthe process of oxidation and/or reduction and the concentration of eachneurochemical. The Cottrell Equation relates to quiet solutionexperiments wherein the potential is instantaneously switched from aninitial value E_(i) to a final potential, then held constant for a fixedtime, then switched back to E_(i). If material diffuses to a planarelectrode surface in only one direction (linear diffusion) then theexact description of the current-time curve is the Cottrell Equation.

Current-time relationships with a circular electrode are defined inelectrochemistry by the Cottrell Equation. For a long time, otherelectrode sizes and experiments using different electrolysis times wereconsidered deviations from the Cottrell Equations that could beconsidered negligible. However, Wightman et al. observed that lineardiffusion is not enough to describe the action that takes place atspherical microelectrodes (Dayton M A et al., 1980, Anal. Chem.52:948-950). The quiet solution behavior of very small electrodes isdifferent and is better described by a steady state equation in whichthe radius of the electrode is taken into account (Adams R N et al.,1982, Handbook of Psychopharmacology, pp. 1-74). This equation issuitable for calculating the edge effect or spherical steady-statecontribution for even a 300-micron diameter electrode. Such acalculation reveals that the edge effect or spherical steady-statecontribution adds approximately 30% current to the linear diffusioncomponent for an electrolysis time of only one second (Dayton M A etal., 1980, Anal. Chem. 52:948-950).

Microvoltammetric circuits using several types of carbon pastemicroelectrodes have been developed and refined (Broderick P A, 1995,U.S. Pat. No. 5,433,710; Broderick P A, 1996, EP 90914306.7; Broderick PA, 1999, U.S. Pat. No. 5,938,903). Reliable separation andquantification of neurotransmitters including norepinephrine, serotonin,and dopamine as well as some of their precursors and metabolites is nowpossible (Broderick P A, 1989, Brain Res. 495:115-121; Broderick P A,1988, Neurosci. Lett. 95:275-280; Broderick P A, 1990, Electroanalysis2:241-245).

One electrode for in vivo electrochemical studies was developed in thelaboratory of Ralph Adams (Kissinger P T et al., 1973, Brain Res.55:209). Using carbon paste electrodes with diameters reaching 1.6 mmand Ag/AgCl (3M NaCl) reference electrodes, neurotransmitters includingdopamine and norepinephrine and their metabolites were detected (notseparated), as a single peak in rat caudate nucleus with finite currentelectrochemistry and cyclic voltammetry.

Extensive refinements to microelectrodes and to in vivo electrochemistryhave been made (Broderick P A, 1990, Electroanalysis 2:241-245). Therecent development of a stearate-carbon paste probe along with anelectrode conditioning process has resulted in reliable separation anddetection of norepinephrine, dopamine, and serotonin (Broderick P A,1996, EP 90914306.7; Broderick P A, 1999, U.S. Pat. No. 5,938,903). Inaddition, other types of microelectrodes with increased sensitivity andreliability continue to be developed (Broderick P A, 1996, EP90914306.7; Broderick P A, 1999, U.S. Pat. No. 5,938,903). Anelectrochemically pre-treated carbon fiber electrode allows thedifferentiation of dopamine from DOPAC (Akiyama R A et al., 1985, AnalChem. 57:1518), as do microelectrodes used in the instant invention.

Previous in vitro analysis techniques have yielded disappointingresults. Prior ex vivo studies attempted to circumvent these problemswith the microdialysis technique (During M J et al., 1993, Lancet341:1607-1610; Lehmann A et al., 1991, Neurotransmitters and Epilepsy,pp. 167-180). Dialysis tubing placed on or within the brain is perfusedwith artificial CSF or Krebs-Ringer bicarbonate solution, and theperfusate is then analyzed with High Performance Liquid Chromatography(HPLC) with electrochemical detection; this provides information aboutthe extracellular environment. However, this technique has beencriticized because of the local gliosis caused by the dialysis probesand the perfusion process that can alter the biochemical parametersunder study. In addition, the perfusate is analyzed outside the brainand therefore in contrast to microvoltammetry measurements are not trulyin situ or in vivo.

Epilepsy is a neurological disorder characterized by transientelectrical disturbances of the brain that may be studied byelectrophysical techniques. Neurotransmitter data from experimentalepilepsy models and in vitro analysis of surgically resected specimensfrom patients with partial epilepsy have thus far yielded conflictingresults. These conflicting results may be due to significant variationsbetween samples as well as choice of controls. Additionally, highlylocalized changes in epileptic cortex are not detectable using wholetissue homogenates. In general, increased activity in noradrenergic,dopaminergic, and serotonergic systems are believed to reduce corticalexcitability and decrease seizure activity (Delgado-Escueta A V, 1984,Ann Neurol. 16(Suppl.): 145-148). However, human temporal lobe epilepsyis a complex disorder that may involve the dysfunction of distinctneuronal systems including the hippocampus and entorhinal cortex, thetemporal neocortex, or combinations of these structures. Therefore, thecontribution of different neurotransmitter systems to epileptogenesis ina given patient likely varies with lesion location and the etiology ofepilepsy. Furthermore, recent studies demonstrating presynapticinhibitory serotonin autoreceptors,-in hippocampus (Schlicker E et al.,1996, Naunyn Schmiedebergs Arch Pharmacol. 354:393-396) and a dual rolefor norepinephrine in epileptogenesis (Radisavljevic Z et al., 1994,International Journal of Developmental Neuroscience 12:353-361) suggestan even more complex situation.

Recent studies are now defining a syndrome of neocortical temporal lobeepilepsy that has distinct clinicopathologic and electrophysiologicfeatures from mesial temporal lobe epilepsy (Pacia S V et al., 1997,Epilepsia 38:642-654; Pacia S V et al., 1996, Ann Neurol 40:724-730).While both mesial temporal lobe epilepsy and neocortical temporal lobeepilepsy are potentially treatable with surgical resection when seizuresare refractory to antiepileptic medication, the type and extent oftemporal lobe resection necessary to achieve a seizure free outcome maydiffer. Neocortical temporal lobe epilepsy patients may requireresections tailored to include the epileptogenic zone. These resectionsmay lie outside the boundaries of a standard temporal lobe resectionperformed for mesial temporal lobe epilepsy. Neurochemistry usingmicrovoltammetry may provide a means for defining the epileptogenic zonein these patients.

Other techniques for detecting neurotransmitters in real time and invivo fall short of the instant invention. These previous methods such asdialysis have limitations such as those described in During M J et al.,1993, Lancet 341:1607-1610; Ferrendelli J A et al., 1986, Adv. Neurol.44:393-400; Goldstein D S et al., 1988, J Neurochem 50:225229; Janusz Wet al., 1989, Neurosci Res 7:144153; Kawaguchi Y et al., 1998, JNeurosci 18:6963-6976.

In vivo detection of neurotransmitters and other chemicals is alsoimportant for diagnosing and treating movement disorders such as spinalcord injuries and brain injuries. Current techniques are limited, inpart, in their relative inability to monitor neural chemistry in realtime in a freely behaving animal or human which may limit theirdiagnostic and/or therapeutic efficacy. Movement may be generated by acentral pattern generator (CPG), i.e. a neuronal network capable ofgenerating a rhythmic pattern of motor activity either in the presenceor absence of phasic sensory input from peripheral receptors.

Central pattern generators have been identified and analyzed in morethan fifty rhythmic motor systems and CPGs can generate a variety ofmotor patterns. A universal characteristic of this wide variety of motorpatterns is that they consist of rhythmic and alternating motions of thebody or appendages. It is the rhythmicity of these behaviors that makethese behaviors appear stereotypic. It is the repetitive quality ofthese behaviors that enables stereotypic behaviors to be controlledautomatically. This automaticity or autoactivity means that there may belittle or no need for intervention from higher brain centers when theenvironment remains stable.

The simplest CPGs contain neurons that are able to burst spontaneously.Such endogenous bursters can drive other motor neurons and some motorneurons are themselves, endogenous bursters. Importantly, bursters arecommon in CPGs that produce continuous rhythmic movement, such aslocomotion. But, locomotion is an episodic, rhythmic behavior and thus,further regulation by neurochemicals becomes necessary. Endogenousbursts (cell firing) of neurons involved in locomotion must be regulatedby neurotransmitters and neuromodulators, i.e., substances that canalter the cellular properties of neurons involved in CPGs. Briefdepolarizations occur and lead to maintained depolarizations (plateaupotentials) that can last for long periods of time. These maintaineddepolarizations far outlast the initial depolarization and it is thesemaintained depolarizations that are necessary for rhythmic movements.The generation of rhythmic motor activity by CPGs can be altered byamines and peptides (Grillner S et al., 1987, Trends Neurosci. 10:34-41;Rossignol S et al., 1994, Curr. Opin. Neurobiol. 4:894-902), therebyenabling a CPG to generate an even greater variety of repetitive motorpatterns. Motor CPGs produce a complex temporal pattern of activation ofdifferent groups of motor functions and each pattern can be divided intoa number of distinct phases even within a phase. CPGs are time-dependent(Pearson K et al., 2000, Principles of Neural Science, 4th edition, pp.738-755).

Serotonin is an important neuromodulator for CPGs and can control theCPG underlying the escape swim response in the mollusc, Tritoniadiomedea. The dorsal swim interneurons (DSIs) are a bilaterallyrepresented set of three 5-HT-ergic neurons that participate in thegeneration of the rhythmic swim motor program. Serotonin from these CPGneurons is said to function as both a fast neurotransmitter and as aslower neuromodulator. In its modulatory role, 5-HT enhances the releaseof neurotransmitter from another CPG neuron, C2 and also increases C2excitability by decreasing spike frequency adaptation. Serotoninintrinsic to the CPG may neuromodulate behavioral sensitization andhabituation. Serotonin intrinsic to the DSI enhances synaptic potentialsevoked by another neuron in the same circuit (Katz P S, 1998, Ann. NYAcad. Sci. 860:181-188; Katz P S et al., 1994, Nature 367:729-731).

In another mollusc, the pteropod Clione limacina, the CPG for swimmingis located in the pedal ganglia and formed by three groups ofinterneurons which are critical for rhythmic activity. The endogenousrhythmic activity of this CPG was enhanced by 5-HT (Arshavsky Y I etal., 1998, Ann. NY Acad. Sci. 860:51-69). In the pond snail, Lymnaeastagnalis, 5-HT is the main neurotransmitter in its stereotypic feedingcircuit (Sadamoto H et al., 1998, Lymnaea Stagnalis. Neurosi. Res.32:57-63). In the sea slug, Aplysia, the CPG for biting is modulatedboth intrinsically and extrinsically. Intrinsic modulation has beenreported to be mediated by cerebral peptide-2 (cp-2) containing CB1-2interneurons and is mimicked by application of CP-2, whereas extrinsicmodulation is mediated by the 5-HT-ergic metacerebral cell (MCC) neuronsand is mimicked by application of 5-HT (Morgan P T et al., 2000, J.Neurophysiol. 84:1186-1193).

In vertebrates, the 5-HT somatodendritic nuclei, the raphe, comprise themost expansive and complex anatomic and neurochemical system in CNS.Raphe nuclei almost exclusively reside along the midline in the rat andin the primate. Fewer reside along the midline, but several exhibit aparamedian organization (Azmitia E C, 1986, Adv. Neurol. 43:493-507).The rostral 5-HT raphe group and caudal linear nucleus sends 5-HTefferents to A₉ basal nuclei motor systems and the caudal 5-HT group,whereas the interfascicular aspect of the 5-HT-ergic dorsal rapheprojects efferents to A₁₀ basal ganglia (nuclei) regions (Jacobs B L etal., 1992, Physiol. Rev. 72:165-229).

Electrophysiological studies have shown that the most prominent actionof increased 5-HT cell firing, in 5-HT somatodendrites in treadmilllocomotion for example, is to increase the flexor and extensor burstamplitude of 5-HT cell firing in dorsal raphe, (DR) somatodendrites for5-HT, during locomotion (Barbeau H et al., 1991, Brain Res.546:250-260). Further evidence for 5-HT controlling motor output is seenfrom studies in which 5-HT, directly injected into the motor nucleus ofthe trigeminal nerve, increased the amplitude of both the tonicelectromyogram of the masseter muscle and the externally elicitedjaw-closure (masseteric) reflex (McCall R B et al., 1979, Brain Res.169:11-27; McCall R B et al., 1980, Eur. J Pharmacol. 65:175-183;Ribeiro-Do-Valle L E et al., 1989, Soc. Neurosci. Abstr. 15:1283). Infact, Jacobs and Azmitia have proposed that 5-HT's primary function inCNS neuronal circuitry is to facilitate motor output (Jacobs B L et al.,1992, Physiol. Rev. 72:165-229).

Serotonin neurons within 5-HT somatodendrites depolarize with suchextraordinary regularity that they exhibit automaticity, i.e., they canact by a CPG and produce plateau potentials. Thus, 5-HT neurons exhibitrepetitive discharge characteristics. Increased 5-HT neuronal cellfiring in somatodendritic raphe nuclei generally precedes the onset ofmovement or even increased muscle tone in arousal by several seconds andis maintained during sustained behavior (Jacobs B L, 1986, NeurochemicalAnalysis of the Conscious Brain: Voltammetry and Push-Pull Perfusion,Ann. NY Acad. Sci., pp. 70-79). Importantly, 5-HT cell firing in raphenuclei is sometimes phase-locked to repetitive behavioral stereotypicresponses. The regular firing of 5-HT somatodendrites in raphe nuclei isactivated preferentially. This activation is associated with locomotionand chewing, stereotypic behaviors that are stimulated by CPGs (Jacobs BL et al., 1991, Pharmacol. Rev. 43:563-578). Serotonin intrinsic CPGshave been reported to be responsible for inducing rhythmic motoractivity in the spinal cord of the turtle and the lamprey (Guertin P Aet al., 1998, Neurosci. Lett. 245:5-8; Harris-Warrick R M et al., 1985,J. Exp. Biol. 116:27-46). The evidence in the lamprey suggests that 5-HTmay have a role in the generation of a family of related undulatorymovements, including, swimming, crawling, and burrowing, by a singleCPG.

In addition to neurological disorders and injuries, the device andmethods of use provided herein may be used for for brain cancerdiagnosis and treatment. Current imaging technology is limited withrespect to tumor visualization in neural tissue. For example, magneticresonance imaging MRI is limited in its ability to detect tumorinfiltration into white matter. This may hinder a physician's ability torender a diagnosis and/or prognosis. It further limits the ability totreat the patient by, for example, hindering a surgeon from definingtumor boundaries to remove the tumor. Alternatively, an inability tovisualize cancerous cells or tissue in white matter may hinder aphysicians ability to monitor the efficacy of a chemotherapy regimen.

SUMMARY OF THE INVENTION

The present invention relates to devices and methods formicrovoltammetric and/or chronoamperometric imaging of temporal changesin neurotransmitter concentrations in living humans and non-humananimals comprising contacting cells with a Broderick probe or BRODERICKPROBE® sensor, applying a potential to said Broderick probe, andgenerating a temporally resolved microvoltammogram. The method mayfurther comprise determining from said microvoltammogram the presenceand concentration of at least one marker selected from the groupconsisting of serotonin, dopamine, ascorbic acid, norepinephrine,γ-aminobutyric acid, glutamate, neurotensin, somatostatin, dynorphin,homovanillic acid, uric acid, tryptophan, tyrosine, nitrous oxide, andnitric oxide. Methods of the invention may further comprise comparingthe microvoltammogram and/or neurotransmitter concentrations to areference or control microvoltammogram and/or neurotransmitterconcentration(s).

The present invention relates to devices and methods for treatingepilepsy. More specifically, the invention relates to the use ofBroderick probes to ascertain neurotransmitter levels in the brains ofpatients having epilepsy, especially temporal lobe epilepsy. In someembodiments of the invention, regions of the brain to be resected areidentified using Broderick probes. In some embodiments of the invention,regions of the brain to be targeted for pharmaceutical therapy areidentified using Broderick probes.

The present invention also relates to devices and methods for reliablydistinguishing temporal lobe gray matter from white matter usingBroderick probes with microvolatammetry.

The invention further relates to methods of brain cancer diagnosis usingdistinct white matter voltammetric signals as detected by Broderickprobes. The invention further relates to diagnosis of other white matterdiseases. Nonlimiting examples of white matter diseases are multiplesclerosis, leukodystrophies, mitochondrial diseases, lipid disorders andglial cell-related disorders whether these glial cells or glia arenormal, abnormal, modified or cultured and the like.

The present invention further relates to devices and methods fordiagnosing and treating cocaine psychomotor stimulant behaviors. In someembodiments of the invention electrodes may be contacted with a subjectto ascertain changes in neurotransmitter levels, e.g. due to releaseand/or reuptake, in real time. In some embodiments, the inventionprovides methods of predicting the occurrence of movement disordereffects of a drug. Nonlimiting examples of movement disorders arecocaine addiction, Huntington's disease, Parkinson's disease, Autism,Lesch-Nyhan Disease and the like.

The present invention further provides devices and methods fordiagnosing pathologies and/or abnormalities of neurotransmitter levels.Neurotransmitters that may be detected by the techniques of theinvention may be selected from the group consisting of serotonin (5-HT),dopamine (DA), ascorbic acid (AA), norepinephrine (NE), γ-aminobutyricacid (GABA), glutamate, neurotensin, somatostatin, dynorphin,homovanillic acid, uric acid (UA), tryptophan, tyrosine, nitrous oxide,and nitric oxide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic diagram of the Broderick probe electrodes for theselective electrochemical detection, in vitro, in vivo, and in situ, ofneurotransmitters, neuromodulators, metabolites, precursors and peptidesin humans and animals, centrally and peripherally. Diagram of electrodeis greatly oversized; actual sizes can range from numbers of nanometersin single digits to a few hundred microns to millimeters.

FIG. 2.A and B. Semiderivative voltammograms from mesial temporal lobeepilepsy patients #8, and neocortical temporal lobe epilepsy patient#14, when the indicator electrode was placed in anterolateral temporalneocortex are shown. The potential applied to the indicator electrode inmillivolts is plotted on the x-axis. The current derived from eachmonoamine is plotted on the y-axis. The electrochemical signals areplotted and the characteristic oxidative potentials for eachneurotransmitter were identified. Mesial temporal lobe epilepsy patient#8 exhibited the norepinephrine and serotonin signature on thevoltammogram at peak oxidation potentials of 0.17V for norepinephrineand 0.305V for serotonin. Neocortical temporal lobe epilepsy patient #14exhibited dopamine and serotonin signals on the voltammogram at peakoxidation potentials of 0.115V for dopamine and 0.295V for serotonin.Data are drawn from the original voltammogram, recorded in situ, i.e.,actual raw data are presented.

FIG. 3.A and B. Microvoltammograms, recorded in vivo in real time ofneurons in the nucleus accumbens (NAcc) of freely moving and behaving,male, Sprague-Dawley laboratory rats. The oxidation potential inmillivolts is plotted on the x-axis. The current derived from eachneurochemical is plotted on the y-axis [Current Scale=25 pAs^(−1/2) 12.5mm^(1/2))]. Panel A was recorded two weeks after surgical implantationof Broderick probes under sodium pentobarbital anesthesia, actually onNov. 30, 1992 and panel B was recorded seven months later on May 11th,1993. Both voltammograms represent endogenous release of dopamine (DA)and serotonin (5-HT) in the basal nucleus, NAcc, while the animal isexhibiting normal motor activity in the open-field behavioral paradigm.Animal was not treated with drugs at either recording time, nor was theanimal treated with drugs during the seven-month period; recordingstaken during the seven-month period were stable as well.

FIG. 4. In situ microvoltammetric recording from hippocampal alveus(white matter in hippocampus, left panel) and neocortex stem (whitematter in neocortex, right panel). Tissue was resected from a mesialtemporal lobe epilepsy patient (Patient #7).

FIG. 5.A and B. (A) Gray Matter recordings (Voltammograms) from resectedtissue from temporal lobe epilepsy patients. Left to Right: Neocortex,Patient 8; Pyramidal Layer, Patient 4; Granular Cells of the DentateGyrus, Patient 3. (B) White Matter recordings (Voltammograms) fromresected tissue from temporal lobe epilepsy patients. Left to Right:Neocortex, Patient 8; Subiculum, Patient 4; Alveus, Patient 3. X-axis:Oxidation potentials in millivolts. Y-axis: Current in picoamperes persemidifferentiation of the second.

FIG. 6. Illustration of Broderick probe operation detectingneurotransmitters along with a resulting microvoltammogram.

FIG. 7. Neurochemistry and Behavior: Line graph depicting endogenous5-HT release (open circles) at basal nucleus, Ag terminals, of neuronsin the dorsal striatum (DStr), detected in real time, while the freelymoving, male, Sprague-Dawley laboratory rat is actually behaving, duringnormal/natural movement (first hour) and subsequent habituation behavior(second hour). Serotonin, detected within seconds of release, is plottedwith a line graph derived from infrared photobeam monitoring of behavior(closed circles): locomotion (ambulations, left panel); stereotypy (finemovements, right panel). Open-field behaviors were studied in units offrequency of events recorded every 100 ms during normal/naturalbehavior. Data show that normal episodic, rhythmic nature of locomotormovement may be neuromodulated by 5-HT within the basal nucleus.

FIG. 8. Neurochemistry and Behavior: Line graph depicting endogenous5-HT release (open circles) at basal nucleus A₁₀ terminals,ventrolateral nucleus accumbens (v1NAcc), in real time, while the freelymoving, male, Sprague-Dawley laboratory rat is actually behaving, duringnormal/natural movement (first hour) and subsequent habituation behavior(second hour). Serotonin, detected within seconds of release, is plottedwith a line graph derived from simultaneous infrared photobeammonitoring of behavior (closed circles): locomotions (ambulations, leftpanel); stereotypy (fine movements, right panel). Open-field behaviorswere studied in units of frequency of events, which were recorded every100 ms during normal/natural locomotor behavior. Data show that normalepisodic, rhythmic nature of locomotor movement may be neuromodulated by5-HT within the basal nucleus, Alo terminals.

FIG. 9. Neurochemistry and Behavior: Line graph depicting endogenous5-HT release (open circles) at basal stem nucleus, DA A 10 terminals,somatodendrites, ventral tegmental area (VTA), in real time, while thefreely moving, male, Sprague-Dawley laboratory rat is actually behaving,during normal/natural movement (first hour) and subsequent habituationbehavior (second hour). Serotonin, detected within seconds of release,is plotted with a line graph derived from simultaneous infraredphotobeam monitoring of behavior (closed circles): locomotion(ambulations, left panel); stereotypy (fine movements, right panel).Open-field behaviors were studied in units of frequency of events, whichwere recorded every 100 ms during normal/natural locomotor behavior.Data show that normal episodic, rhythmic nature of locomotor movement isexhibited and can be detected with this biotechnology. However, still,very rhythmic, 5-HT neuromodulation of movement in VTA exhibits adifferent pattern of rhythm with movement than that pattern, seen inbasal nuclei.

FIG. 10. Cocaine Neurochemistry and Behavior: Line graph depictingendogenous 5-HT release (open circles) at basal nucleus A₁₀ terminals,v1NAcc, in real time, while the freely moving, male, Sprague-Dawleylaboratory rat is actually behaving, during cocaine-induced behavior(intraperitoneal injection of cocaine: two hour study). Serotonin,detected within seconds of release, is plotted with a line graph derivedfrom simultaneous infrared photobeam monitoring of behavior (closedcircles): locomotions (ambulations, left panel); stereotypy (finemovements, right panel). Open-field behaviors were studied in units offrequency of events, which were recorded every 100 ms duringnormal/natural locomotor behavior. Data show that cocaine disrupted thenormal episodic, rhythmic nature of locomotor and stereotypic movementwhich may be neuromodulated by 5-HT within the basal nucleus, Aloterminals. Data suggest that cocaine caused neuroadaptive process in5-HT mechanisms in DA basal nuclei.

FIG. 11. Cocaine Neurochemistry and Behavior: Line graph depictingendogenous 5-HT release (open circles) at basal stem nucleus, DA A 10somatodendrites, VTA, in real time, while the freely moving, male,Sprague-Dawley laboratory rat is actually behaving, during cocainebehavior (subcutaneous injection of cocaine: four hour study).Serotonin, detected within seconds of release, is plotted with a linegraph derived from simultaneous infrared photobeam monitoring ofbehavior (closed circles): locomotion (ambulations, left panel);stereotypy (fine movements, right panel). Open-field behaviors werestudied in units of frequency of events, which were recorded every 100ms during normal/natural locomotor behavior. Data show that cocainedisrupted the normal episodic, rhythmic nature of locomotor movement,likely by disturbing 5-HT neuromodulation of behavior in DA motorcircuits and causing neuroadaptation.

FIG. 12.A and B. Schematic of Broderick probes in a grid format on thebrain (A) and side view (B).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an electrode or sensor having a constructionand an indicator, where the sensor may be used for detectingbiomolecules, including but not limited to, for example, but not limitedto, neurotransmitters, drugs, amyloid proteins, stabilizing proteins,and chemicals, in order to diagnose or determine a diseased state orcondition.

In one embodiment, the Broderick probe is a microelectrode comprisinggraphite, oil, and a material selected from the group consisting ofglycolipids, lipoproteins, saturated and unsaturated fatty acids, andperfluorosulfonated materials.

In some embodiments of the invention, the Broderick probe is a Broderickprobe microelectrode having an indicator. Within the field ofelectrochemistry, this sensor may be termed the indicator microelectrodeand may also be called the working microelectrode. The surface of themicroelectrode consists of carbon-base and is the electrochemicaldevice.

Broderick probes do not promote bacterial growth either before or aftersterilization with gamma irradiation. Gamma irradiation treatment wasperformed by Sterigenics International, Inc., Haw River, N.C.

A non-limiting example of a Broderick probe or sensor comprising aconstruction and an indicator as depicted in FIG. 1. Further details andexamples may be found in U.S. Pat. Nos. 4,883,057, 5,443,710, and5,938,903, all to P. A. Broderick including circuit diagrams and methodsof making Broderick probes. Broderick probes may be in electricalcontact with an auxiliary electrode and/or a reference electrode. Itwill be apparent to those of ordinary skill in the art, particularly inview of the cited patent documents, that “Broderick probe” is a termthat relates to a number of electrodes that vary by composition and thetype of circuit in which it is employed and that these variations giverise to differences in detection properties.

One means for electrochemically measuring the concentration of one ormore biomolecules or markers include, but are not limited to, circuitsof square wave, amperometric semidifferential/semiderivative,voltammetric, linear scan, differential pulse, double differntial pulse,and, chronoamperometric circuits. The auxiliary microelectrode,reference microelectrode, and inventive sensor are functionallyconnected to the means for electrochemical measurements.

Broderick probes are miniature carbon-based sensors that are able todetect electrochemical signals for a vast number of neurotransmitters,neuromodulators and metabolites, including neuropeptides, hormones,vitamins, and the like (Broderick P A, 1989, U.S. Pat. No. 4,883,057;Broderick P A, 1995, U.S. Pat. No. 5,433,710; Broderick P A, 1997, EP0487647 B1; Broderick P A, 1999, U.S. Pat. No. 5,938,903; Broderick P A,1999, Hong Kong, HK #1007350). These probes have made it possible toroutinely and selectively detect in discrete neuroanatomic substrates ofliving human and animal brain, the monoamines, DA, NE, and 5-HT, inaddition to the precursor to 5-HT, 1-tryptophan (1-TP), ascorbic acid(AA) and uric acid (UA) (Broderick P A, 1988, Neurosci. Lett.95:275-280; Broderick P A, 1989, Brain Res. 495:115-121; Broderick P A,1990, Electroanalysis 2:241-251; Broderick P A, 2000, Epilepsia41(Suppl.):91; Broderick P A et al., 2000, Brain Res. 878:49-63). It isalso possible to differentiate catecholamines, DA and NE,electrochemically using these probes (Broderick P A, 1988, Neurosci.Lett. 95:275-280; Broderick P A, 1989, Brain Res. 495:115-121; BroderickP A, 1990, Electroanalysis 2:241-251; Broderick P A, 2000, Epilepsia41(Suppl.):91; Broderick P A et al., 2000, Brain Res. 878:49-63). Morerecently, it has been found that these probes are also capable ofelectrochemical detection of somatostatin and dynorphin A (Broderick PA, 2000, Epilepsia 41(Suppl.):91).

The Broderick probe, electrode, BRODERICK PROBE® sensor, or sensor, asused herein, may be prepared in a wide range of sizes and formulationseach with different detection capabilities. In particular, oneembodiment of the Broderick comprises a construction and indicatorportion. The Broderick probe or sensor is useful for intraoperativerecordings and continuous recordings with data logging and telemetry.

The sensor is partially or fully encased in an encasement, where theencasement is made of a conducting, semi-conducting, or non-reactivematerial, such as for example, metals, polymers, or blends thereof, forexample, but not limited to, polytetrafluoroethylene, fluorinatedethylene-propylene, perfluoroalkoxy polymer resin,polymethylmethacrylate, polyethylethacrylate, steel, stainless steel,silicon, germanium, silver, platinum, gold, or combinations thereof. Theshape of the encasement may be a hollow three-dimensional surface, wherethere is an opening on either or both ends of the sensor which allowscontact of the indicatorwith the neural cells, body, blood, or urine.The shape of the encasement, and thereby sensor, has an end face in theshape of any geometric shape. By end face is meant the ends of theencasement. For example, if the encasement is in the shape of a hollowcylinder, then the circle faces are the end faces of the sensor, seeFIG. 1. Non-limiting examples of geometric shapes include: a circle, atriangle, a quadrilateral, a rhombus, a parallelogram, a rectangle, or apolygon of any number of sides, pentagonal, hexagonal, or octagonal.

The encasement may be an inverted well or the like, having the inventivesensor material. The partially or fully encased sensor may be, but notlimited to, flat, horizontal or vertical. The sensor material may befitted into, for example, an electroencephalographic (EEG) grid, strip,or depth electrodes for recordings for ictal, interictal, intracranialimaging, for image-guided surgery and gamma-knife surgery in epilepsy ortumor patients, and the like. Sensors may also, for example, be fittedinto stimulating electrodes for thalamic and globus pallidus recordingsand the like in Parkinson's patients.

The construction portion of the sensor has a conducting and/orsemi-conducting material, including for example, a conducting metal orsemi-conducting metal, or combinations thereof. Non-limiting examples ofthe construction include: steel, stainless steel, silicon, germanium,silver, platinum, or gold, or combinations thereof.

The indicator portion of the sensor has at least one form of carbon, orcombinations thereof, and at least one lipid or entity having a lipid,or combinations thereof. Carbon comes in several forms, including butnot limited to, graphite, fullerenes, cylindrical fullerenes,buckminsterfullerenes, buckyballs, nanotubes, probingtubes, cold formcarbon steel, white carbons, dioxosilane, diamonds, or combinationsthereof. A lipid is any of a group of organic compounds, including fats,oils, waxes, sterols, and triglycerides, that are insoluble in water butsoluble in non-polar organic solvents. Non-limiting examples of lipids,or entities having lipids, include fats, oils, animal fats or oils,plant fats or oils, mineral oils, nujol oil, glycerol containing lipids,membrane lipids, soaps or detergents, waxes, cells, cell components,stem cells, electroplaques, lipoproteins, fatty acids, glycerides,monoglycerides, diglycerides, triglycerides, artificial or synthesizedfats or oils, heifer fats, ox-depot fats, Valeria indica fats, tallow,red tallow, Malabar tallow, vegetable tallow, cocoa butter, soybean oil,safflower oil, sesame oil, peanut oil, coconut oil, linoleic acid,linoleic acid in vegetable oil, soybean oil, cottonseed oil, corn oil,or poppyseed oil, lauric acid, lauric acid in coconut oil, cholesterol,phosphotidylcholine, phosphotidylethanolamine, sphingomyelin, lecithin,lysolecithin, steroids, isoprenoids, eicosenoids, sodium alkyl benzenesulfonate, sodium lauryl sulfate, jejoba wax comprised of gadoleic acid,N-stearoylcerebroside, N-stearoylsphingosine, cardiolipin, orcombinations thereof.

In one embodiment of the invention, the indicator may be pre-treated,inserted, or coated with a biomolecule, or combinations thereof.Biomolecules include, but are not limited to, pharmaceutical compounds,pharmaceutical compounds specific for neurodegenerative orneuropsychiatric diseases, disorders, and conditions, neurotransmitters,neuromodulators, hormones, surfactants, soaps, detergents, pramipexole,topiramate, clozapine, dopamine, serotonin, norepinephrine,acetylcholine, adenosine, estrogen, vitamins, vitamin A, vitamin E,brain lipids, phosphotidylethanolamine, tallow, sodium lauryl sulfate,N-acetyl-aspartate, choline, lactate, uric acid, stabilizing proteins,amyloid proteins, ascorbic acid, γ-aminobutyric acid, glutamate,neurotensin, somatostatin, dynorphin, homovanillic acid, nucleic acids,tryptophan, tyrosine, nitrous oxide, nitric oxide, surfactants, orcombinations thereof.

Non-limiting examples of sensor formulations include:

a) any form of carbon, fats and oils, with or without pre-treatment witha pharmaceutical compound for neurodegenerative and neuropsychiatricdisease such as but not limited to pramipexole, topiramate andclozapine, or combinations thereof;

b) any form of carbon, fats and oils, with and without pre-treatmentwith neurotransmitters, neuromodulators, nucleic acids, hormones andvitamins, such as but not limited to dopamine, serotonim,norepinephrine, acetylcholine, adenosine, estrogen, vitamin A andvitamin E, or combinations thereof.

c) any form of carbon, fats and oils, with and without pre-treatmentwith surfactants, such as but not limited to, brain lipids, such asphosphotidylethanolamine and soaps and detergents, such as but notlimited to, tallow and sodium lauryl sulfate, or combinations thereof.

d) graphite, lipids, fatty acids and nujol oil, with and withoutpre-treatment with biomolecules, such as those listed in a) through c)above.

e) graphite, lipids or fatty acids, and nujol oil, with or withoutpre-treatment with biomolecules, such as those listed in a) through c)above.

f) graphite, lipids, fatty acids, and nujol oil, with a coating and/orinsertion of biomolecules within the inventive sensor, including suchbiomolecules listed in a) through c) above.

g) carbon or allotropes of carbon, an entity containing a lipid, wherethe entity includes but is not limited to cells, components of cells,stem cells, electroplaques, and lipoproteins, with and withoutpre-treatment with and/or insertion of biomolecules biomolecules withinthe inventive sensor, including such biomolecules listed in a) throughc) above.

The sensors of the invention may be used in any or all methods describedherein. In some embodiments, the size of the sensor is from less thanabout 1 nanometer to about 10 millimeters in width, and from less thanabout 1 nanometer to about 10 millimeters in length. The indicatorportion having some form of carbon may be present in a useful amountranging from about 1 microgram to about 100 grams, while the lipid whichis incorporated, coated, or inserted in the carbon, is in an amount fromabout 1 microgram to about 100 grams.

In some embodiments, the following formulations of the indicator portionmay be used in a sensor having an end face diameter of about 150micrometers. FORMULATION OTHER COMPONENTS AMOUNTS A Graphite 1.5 g Nujoloil 1.24 g Lauric acid 100 mg B Graphite 0.075 g Nujol oil 0.62 gN-Stearoylcerebroside 5 mg C Graphite 0.075 g Nujol oil 0.62 gN-Stearoylsphingosine 5 mg D Graphite 0.30 g Nujol oil 0.25 gCardiolipin 20 mg

A critical element for sensor composition, in particular, the indicatorportion, is the carbon. Carbon is found free in nature in threeallotropic forms, amorphous, graphite and diamond. There are variousforms of carbon, including but not limited to, graphite, fullerenes,cylindrical fullerenes, buckminsterfullerenes, buckyballs, nanotubes,probingtubes, cold form carbon steel, white carbons, dioxosilane,diamonds, or combinations thereof. Carbon is essential to detect otherbioelements and biomolecules which diagnose and treat neurodegenerativeand neuropsychiatric diseases, disorders, or diseases, such as, but notlimited to pramipexole for Parkinson's disease, topiramate for epilepsy,and clozapine for organic psychosis, as well as for detecting anybiomolecules in the body, for example, in blood or urine.

Because of carbon's unique bonding properties, millions of differentorganic chemicals exist in plants, and animals. Covalently bonded carbonatoms form the backbone of organic compounds; carbon has tremendousbonding versatility. Different conformations and different bond lengthsare possible. Solid and semi-solid matter comprised of carbon, arecells, components of cells, such as but not limited to mitochondria andmembranes and proteins, such as but not limited to amyloid proteins andtheir subtypes such as beta amyloid proteins. Three dimensional shapesand chiral and stereoisomers of carbon are important for biological andbiochemical function. Cells and components of cells are comprised of andare reliable sources of, not only of carbon but also, fats, fatty acids,lipids, phospholipids, lipoproteins, neurotransmitters, neuromodulatorsand the like.

The bioelement, carbon, in its different forms, is critical to thematerial comprised in the indicator portion of the inventive sensorbecause of its excellent reducing agent property. It is used as such inpurifying metals in electrodes and in electrical devices, steel tracerresearch, in chemical dating of jewelry, and in cutting glass. Also,critical to the use of carbon in the instant inventive sensor material,is the property of carbon which allows movement of electrons and thusconducts electricity and carbon is a semi-conductor as well. Carbon'sadsorptive properties, such as in wastewater treatment systems, fuel andmining systems, automotive exhaust systems and it is adsorptive in theprocess of removing contaminants from drinking water. Carbon enables asubstance to attach to the surface of another substance. Carbon comes inseveral forms, including but not limited to, graphite, fullerenes,cylindrical fullerenes, buckminsterfullerenes, buckyballs, nanotubes,probingtubes, cold form carbon steel, white carbons, dioxosilane,diamonds, or combinations thereof.

The indicator portions of the inventive sensors also have some type oflipid, fat, or oil. These are water-insoluble substances of plant,animal, or mineral origin, which are mainly comprised of glycerol estersof fatty acids or glycerides. Lipids are greasy oily substances whichalso contain glycerol. The word, “fat” is commonly used to refer totriglycerides that are solid or semi-solid at ordinary temperatures; theword “oil” connotes triglycerides in the liquid phase. The terminologies“fat” and “oil” are often interchanged. Therefore, a triglyceride is acondensation product of one molecule of glycerol and three molecules offatty acid. A mixed triglyceride contains two different fatty acids andhas four isomeric forms. Monoglycerides and diglycerides contain onlyone or two fatty acids respectively and consequently have two or onefree hydroxyl groups. Monoglycerides and diglycerides do not occurnaturally in appreciable quantities except in fats that have undergonepartial hydrolysis. Animal and vegetable fats can have similarcomposition and thus, we must realize the importance of glyceridecomposition in determining the physical properties of fats and oils.

A lipid is any of a group of organic compounds, including fats, oils,waxes, sterols, and triglycerides, that are insoluble in water butsoluble in non-polar organic solvents. Non-limiting examples of lipids,entities comprising lipid, fats, or oils useful in the inventive sensorsinclude animal fats or oils, plant fats or oils, mineral oils, nujoloil, glycerol containing lipids, membrane lipids, soaps or detergents,waxes, cells, cell components, stem cells, electroplaques, lipoproteins,fatty acids, glycerides, monoglycerides, diglycerides, triglycerides,artificial or synthesized fats or oils, heifer fats, ox-depot fats,Valeria indica fats, tallow, red tallow, Malabar tallow, vegetabletallow, cocoa butter, soybean oil, safflower oil, sesame oil, peanutoil, coconut oil, linoleic acid, linoleic acid in vegetable oil, soybeanoil, cottonseed oil, corn oil, or poppyseed oil, lauric acid, lauricacid in coconut oil, cholesterol, phosphotidylcholine,phosphotidylethanolamine, sphingomyelin, lecithin, lysolecithin,steroids, isoprenoids, eicosenoids, sodium alkyl benzene sulfonate,sodium lauryl sulfate, jejoba wax comprised of gadoleic acid,N-stearoylcerebroside, N-stearoylsphingosine, cardiolipin, orcombinations thereof.

Surfactants generally act to lower surface tension. Surfactants arecomposed of relatively large molecules which contain widely separatedgroups of a dissimilar nature such as is seen with the biomolecules.They are generally organic compounds that are amphipathic, and aretypically sparingly soluble in both organic solvents and water.Surfactants are useful for their capability to reduce surface tension aswell as to assist the migration of and to orient molecules orbiomolecules.

The diversity of the charged parts (indicator and/or constructionportions) of the instant inventive sensor allow for versatility in thecharge-transfer or electron-transfer mechanisms of the instant inventivesensors. The versatility in the electron-transfer mechanism allows forversatility in current production, thereby causing not only enhancedsignaling in brain, body fluids, and biomolecules but also allowingdifferent degrees of electron-transfer, which directly correlates todifferent concentrations of biomolecules according to the CottrellEquation. Even the type of lipid, e.g., provides versatility in theelectron-transfer process. For example, the instant inventive sensor(indicator and/or construction portions) which is coated with the lipid,phosphotidylethanolamine, produces significantly enhanced signals forthe biomolecules. Low energy surface of the interfacial regions (or endface), a smaller angle of contact at the end face, and greater migrationof molecules into an adjacent liquid phase all contribute to the noveland unexpected enhanced signaling mechanisms using surfactants in theinstant inventive sensor.

Furthermore, the ability of surface-active biomolecules and/orsurfactants to migrate to the interface of a solution and therefore forman oriented or adsorbed film biomolecules and/or surfactants may be oneof several key properties for the mechanism of action of the instantinventions.

Adsorption is the ability of a molecule to attach to, for example, thesurface of the instant inventive sensor. Adsorption at the surface ofthe instant inventive sensor provides an unexpected and surprisingmemory mechanism, which may be a reflection of a particular biomolecule,for example, but not limited to, neurotransmitters, neuromodulatorsand/or particular pharmaceutical compounds used to treatneurodegenerative and neuropsychiatric disorders, and the like. Thereflection of the biomolecules may occur whether or not pretreatment ispart of the empirical protocol, as the sensor can be placed in contactwith the molecule(s) one or more times so that the consistency of thespecificity of the electron transfer (charge transfer) process willproduce the image.

Without wishing to be bound by theory, between the surface of theinstant inventive sensor and the biomolecule per se, weak covalent bondsare formed by adsorption of the biomolecule onto the surface of theinstant inventive sensor, which is also comprised of adsorptive carbonor carbon allotropes. Although weak covalent bonding may provide arationale for the memory or reflective image of the biomolecule producedby the instant inventive sensor, it is plausible that a number of otherinteractions may occur between the biomolecule and the instantinventions. Chemisorption is an adsorptive interaction between amolecule and a surface in which electron density is shared by theadsorbed molecule or molecules and the sensor surface. Physisorption isanother plausible explanation for the reflective adsorptive image on theinstant inventions. In this mechanism, a biomolecule does not actuallyundergo a specific chemical bonding. Physisorption involves anelectrostatic attraction or dipole-dipole interaction but no realchemical bonds are made. Instead, there is a sharing of electronsbetween the biomolecule and the instant inventive sensor material, thatis to say, as the adsorbate, i.e., the biomolecule migrates closer tothe surface of the sensor, the dipole moment of the adsorbate induces animage dipole. Since dipoles attract each other and generate anelectromagnetic field on the surface, the electromagnetic field mayfactor into reflection or imaging of the biomolecule or biomolecules onthe part of the instant sensor invention.

Other possible explanations are provided by interactions which occur inunsaturated fatty acids which have double or triple bonds. In thisinteraction, memory or reflective image may occur in the instantinventive sensor by the formation of pie bonds. Such a pie bondinteraction may occur between the surface of the instant inventivesensor and biomolecules which are aromatic, benzene-like and moleculeswhich contain a phenyl or phenyl groups such as those which are cited inthe embodiment of the instant inventive sensors. Pi-bonds may accountfor the detection of straight chain amino acid compounds, such asacetylcholine, and benzene-like compounds, such as monoamines, with theinstant inventive sensors. (Adamson, A. W., Textbook of PhysiacalChemistry, Academic Press, New York, 1973; Kissinger, P.T., Heineman, W.R., (Editors) Laboratory Techniques in Electroanalytic Chemistry, SecondEdition, Marcel Dekker, Inc. New York, 1996; Kamat, P. V., Asmus, K. D.,“What's all the excitement about?” Interface, The ElectrochemicalSociety, Volume 5 (number 1), 1996; Dohnalek, Z., Kim, H., Bondarchuk,O., White, J. M., Kay, B. D., Physisorption of N₂,O₂,and CO on fullyoxidized TiO₂ (110) J. Phys. Chem. B Condens Matter Mater SurfInterfaces Biophys. 110 (12): 6629-6635, 2006; Mattil, K. F., Norris,F.A., Stirton, A. J., Swern, D., Bailey's Industrial Oil and FatProducts, (Edited by Swern, D.) Interscience Publishers, a Division ofJohn Wiley & Sons, Inc., Third Edition, 1964; and Hui, Y. H. (Editor),Bailey's Industrial Oil and Fat Products, Edible Oil and Fat Products:Oil and Oil Seeds, A Wiley-Interscience Publication, Volume 2, FifthEdition, 1996, all of which are incorporated herein by reference).

In one embodiment, the sensor may be in the form of a miniaturestainless steel flat, circular disk construction in which the indicatorcomprises graphite incorporated or coated with cerebrosides, stearoyl,lauric acid, or the like, or combinations thereof, and the indicator isfurther incorporated or coated with biomolecules including but are notlimited to, pharmaceutical compounds, pharmaceutical compounds specificfor neurodegenerative or neuropsychiatric diseases, disorders, andconditions, neurotransmitters, neuromodulators, hormones, surfactants,soaps, detergents, pramipexole, topiramate, clozapine, dopamine,serotonin, norepinephrine, acetylcholine, adenosine, estrogen, vitamins,vitamin A, vitamin E, brain lipids, phosphotidylethanolamine, tallow,sodium lauryl sulfate, N-acetyl-aspartate, choline, lactate, uric acid,stabilizing proteins, amyloid proteins, ascorbic acid, γ-aminobutyricacid, glutamate, neurotensin, somatostatin, dynorphin, homovanillicacid, nucleic acids, tryptophan, tyrosine, nitrous oxide, nitric oxide,or combinations thereof, are completely or partially encased inpolytetrafluoroethylene. The sensor may either exist alone or act as apartner to the electroencephalograph grids (EEG) for an epilepsy and/ortumor patient, where a reference and auxiliary electrode may also bepresent.

Another embodiment relates to a 2- or 3-sensor assembly, enclosed in abiocompatible plastic “carry on cap” which has the ability to be removedand reinserted into specific parts or locations of patient brains. Theintracranial recordings recorded using the inventive sensors in a 2- or3-sensor assembly significantly depart from conventional means in thatneurochemicals and neuronal firing rates are determined simultaneously.Also, accompanying devices work, hand in hand, with sensors, haveseveral sets of operational amplifiers which provide mapping of severalneuroanatomic sites simultaneously. However, one skilled in the artwould understand that separate sensors that are not grouped in a sensorassembly may be just as easily be used to obtain recordings.Furthermore, the inventive sensors as described herein may be used inconjunction with other sensors, such as, but not limited to referenceelectroodes

Broderick probes can be used effectively for different applications inhuman and animal surgery, as well as a diagnostic tool for testing, forexample, blood, urine and/or various regions of the body. Preliminarystudies with Broderick probe stearic versus lauric acid electrodes invitro, in situ, and in vivo showed a possible advantage for the lauricacid electrodes for use short-term, e.g., intraoperative recordings, anda possible advantage for stearic acid for use long-term, e.g., chronicmonitoring in humans and animals (Broderick P A, 1989, U.S. Pat. No.4,883,057; Broderick P A, 1995, U.S. Pat. No. 5,433,710; Broderick P A,1997, EP 0487647 B1; Broderick P A, 1999, U.S. Pat. No. 5,938,903;Broderick P A, 1999, Hong Kong, HK #1007350; Hope O et al., 1995,Cocaine has remarkable nucleus accumbens effects on line, with behaviorin the serotonin-deficient Fawn Hooded rat. NIH/NIGMS Symposium,Washington, D.C.).

Broderick probes can detect basal (normal, natural, endogenous or steadystate) concentrations of neurotransmitters and other neurochemicals invivo, in situ and in vitro. They can also detect alterations in theseneurotransmitters or neurochemicals in brain, or body before and afterpharmacological manipulation with drugs or other compounds.Neurochemicals during actual, induced or even mimicked brain diseasescan be detected as well. Example 5 focuses on 5-HT alterations in NAccin freely moving animals during normal open-field behaviors of locomotor(exploratory) and stereotypy compared with, in the same animal, cocainepsychomotor stimulant effects on 5-HT and behavior.

Changing the surface of the sensor changes the capacitance of thesurface of the sensor. The surface of the indicator electrode is acapacitance diffuse double layer (C_(dt)) that allows potential toaccumulate on its surface. Capacitance is a critical aspect of charging(background) current. Charging current is a current pulse that flowsthrough the C_(dt) to allow faradaic electron transfer to begin.Accumulation of potential on the surface of the indicator electrode isnecessary for faradaic electron transfer. Charging current isproportional to electrode surface area; therefore, these miniaturesensors (200 microns and less in diameter) minimize charging currenteffects.

Broderick probes or sensors can be used in conjunction with classicalelectrical circuits used in electrochemistry such as chronoamperometry,differential pulse voltammetry and double differential voltammetry.Another electrical circuit for providing an output signal having amathematical relationship in operation to an input signal can besemiderivative or semidifferential. These two terms are usedinterchangeably here, although these two circuits have some technicaldifferences. Semiderivative electroanalysis diminishes non-faradaiccurrent by the addition of analysis time. In the present studies, a CV37 detector (BAS, West Lafayette, Ind.) was equipped with asemiderivative circuit. This circuit uses a linear scanning methodologyas its basis. Semiderivative treatment of voltammetric data means thatthe signals are recorded mathematically as the first half derivative ofthe linear analog signal. A semiderivative circuit combines anadditional series of resistors and capacitors, called a “ladder network”(Oldham, K, 1973, Anal. Chem. 45:39-50) with the traditional linearscanning technology which then allows more clearly defined waveforms andpeak amplitudes of electrochemical signals than was previously possiblewith linear scanning methodology.

Broderick probe microvoltammograms may be plotted as current versus timeor as current versus applied potential. Other renderings are alsopossible. The concentration of biogenic amines and other materials maybe deduced from these microvoltammograms, e.g. according to the Cottrellequation. According to the invention, a microvoltammogram is broadlydefined as any rendering of the signals from a Broderick probesusceptible to human perception including, but not limited to, paper,electronic, and virtual representations of the Broderick probe signal.An individual of sufficient skill in the art to perceive a Broderickprobe signal in real-time, e.g. from a visual display screen, is alsowithin the contemplation and scope of this definition.

The main strength of in vivo microvoltammetry (electrochemistry) is thatit allows the study of the neurochemical time course of action of normalneurochemistry, as well as the neurochemistry after an administered drugregimen. Temporal resolution is fast, in seconds and milliseconds.Moreover, the attendant microspatial resolution is superior(availability of discrete areas of brain without disruption). Bothhighly sensitive temporal and spatial resolution makes these studiesultimately most efficient for mechanism of action studies Anotherstrength lies in the fact that these in vivo microvoltammetric studiesare done in the freely moving and behaving animal model, using the sameanimal as its own control (studies in the living human brain areunderway as well). Thus, a direct determination of whether or not aneurochemical effect is abnormal can be made because the normalneurochemical effect is seen a priori.

The basic in vivo electrochemistry experiment involves the implantationof an indicator electrode or sensor in a discrete and specified regionof brain, the application of a potential to that electrode or sensor,the oxidation or reduction of the selected neurochemical or biomoleculeand the recording of the resultant current. In essence, the potential isapplied between the indicator or sensor, and the reference electrode;the reference electrode provides a relative zero potential to sense theamount of current produced by the flow of electrons from the biomoleculethrough the indicator. This is an electrochemical technique with whichinformation about an analyte, a neurotransmitter, or its metabolite,including its concentration, is derived from an electrochemical currentas a function of a potential difference. This potential difference isapplied to the surface of an electrochemical electrode. Additionally, anauxiliary electrode provides an electrical ground.

In microvoltammetry, each neurotransmitter, metabolite, precursor toneurotransmitter, etc. is identified by the peak oxidation potential, orhalf-wave potential at which the neurochemical generates its maximumcurrent. Using the Broderick Probe stearic acid electrode inserted inNAcc, the oxidation potential at which DA generates its maximum currentin vivo (physiological pH, 37.5° C.) was empirically determined to be+0.140 V (SE±0.015 V) in over one thousand studies. The oxidationpotential at which serotonin generates its maximum current under thesame conditions was empirically determined to be +0.290 V (SE±0.015 V)in over one thousand studies.

What matters in microvoltammetry is that each of these biogenic amineshave amine groups that are protonated at neutral pH and therefore, existas cations, whereas metabolites of the monoamines are deprotonated atneutral pH and exist as anions (Coury L A et al., 1989, Biotechnology11:1-37). Thus, the monoamine metabolites such as the metabolites of DA,3,4 dihydroxyphenylacetic acid, (DOPAC), 3,4-dihydroxyphenylglycol(DHPG-DOPEG) and homovanillic acid (HVA) cannot interfere with thedetection of DA at the same peak oxidation potential or half-wavepotential, characteristic for DA.

The same principles are applicable to detection of the biogenic amine,5-HT. Serotonin is detected without interference at the same oxidationpotential or half-wave potential from either its metabolite,5-hydroxyindoleacetic acid (5-HIAA) or UA, which is a constituent ofbrain with similar electroactive properties to those of 5-HT. Factorssuch as the significantly lower sensitivity of the indicator electrodeto anions, the charge and diffusion characteristics of eachcatecholamine or indoleamine vis—vis its metabolites, preclude suchinterference. Descriptions of each neurochemical detected by thisinventor with Broderick probes are published in detail (Broderick P A,1995, U.S. Pat. No. 5,433,710; Broderick P A, 1996, EP 90914306.7;Broderick P A, 1999, U.S. Pat. No. 5,938,903; Broderick P A, 1989, BrainRes. 495:115-121; Broderick P A, 1988, Neurosci. Lett. 95:275-280;Broderick P A, 1990, Electroanalysis 2:241-245; Broderick P A, 1993,Pharmacol. Biochem. Behav. 46:973-984; Broderick P A, 2002, Handbook ofNeurotoxicology, Vol. 2, Chapter 13; Broderick P A et al., 2000, BrainRes. 878:48-63; Broderick P A et al., 1997, Neuroscience andBiobehavioral Reviews 21(3):227-260; Broderick P A, 1989, U.S. Pat. No.4,883,057; Broderick P A, 1997, EP 0487647 B1; Broderick P A, 1999, HongKong, HK #1007350; Broderick P A, 2000, Epilepsia 41(Suppl.):91).

An important distinction between the detection of signals inmicrovoltammetry as compared with the detection of signals inmicrodialysis is that in microvoltammetry, the indicator electrode isthe detecting device, whereas in microdialysis methods, the dialysismembrane is a membrane and not the detecting device. The microdialysismembrane is simply a membrane through which perfusate is collected. Theperfusate is then brought to the high performance liquid chromatography(HPLC) device, equipped with an electrochemical column that is theactual detecting device. These electrochemical columns range inmillimeters in diameter, whereas microvoltammetry indicator electrodesrange from single digit microns to a few hundred microns in diameter.

A common misconception is that a microdialysis membrane is a detectingdevice which, in turn leads, incorrectly, to direct comparisons betweenmicrodialysis membranes and microvoltammetry indicator detectingdevices. Whether or not microdialysis membranes are the same size asvoltammetry electrodes is irrelevant because the microdialysis membraneis not the detection technology. Microdialysis membranes simply collectperfusate from brain and this perfusate is then analyzed by HPLC.

Dialysis is a technique based on semipermeability of a collectionmembrane and is not, itself, a detection technique. Existing methods ofdetecting glutamate by microdialysis followed by HPLC andelectrochemical (EC) detection, actually detect a derivative ofglutamate rather than glutamate itself. Similarly, microdialysis methodsof detecting the neurotransmitter acetylcholine are based on detectinghydrogen peroxide, not acetylcholine itself (Stoecker P W et al., 1990,Selective Electrode Rev. 12:137-160). Moreover, correlation between thederivative of glutamate or H₂O₂ detected and the Cottrell Equation hasnever been addressed. Therefore, detection of straight chain carboncompounds by the microdialysis membrane method may be questionable.Broderick probes offer an attractive alternative since they may be ableto directly detect glutamate or acetylcholine.

Generally, quantitation of neurochemistry is described as a percentageof a few data points, over hours, used as “control” in microdialysisstudies. However, Broderick probes are easily calibrated andconcentrations are interpolated from calibration curves (Broderick P Aet al., 2000, Brain Res. 878:49-63).

In one embodiment of the invention, the inventive sensors may be used inneurosurgical procedures. These procedures are guided by a surgeon'svisual inspection of the brain using his/her knowledge of neuroanatomycorrelated with imaging studies generally performed prior to surgery orintraoperatively. The location of the tip of a surgeon's instrumentthrough a three dimensional space co-registration of the surfacecoordinates on the patient's head identified on a preoperative imagingstudy assist in guiding the surgeon. The three-dimensional spaceco-registration can then be co-registered to a three space coordinatesystem on the surgeon's instrument.

Image-guided neurosurgery involves focus localization and is differentfrom the conventional interictal and ictal scalp and intracranialelectroencephalographic (EEG) recordings. Focus localization withimage-guided neurosurgery involves EEG recordings obtained andtopographically characterized. Using the image directed system, the EEGelectrode position is correlated physiologically and anatomically withthe surgical field. Augmented intraoperative localization and surgicaltreatment of, for example, epilepsy and tumors by interactiveimage-guided technology, integrates the neuropsychological,electrophysiological and anatomical data to the surgical field.

One of the advantages of using Broderick probes with microvoltammetry isthat microvoltammograms may be obtained from freely moving and behavingliving animals and humans. Thus, in some embodiments of the inventionanother parameter may be monitored and/or recorded. For example, aBroderick probe microvoltammogram may be acquired from a subject whilesimultaneously monitoring and/or recording the subject's movements (e.g.ambulations and/or fine motor movements). Other examples of parametersthat may be monitored and/or recorded include, inter alia, the presenceand concentration of a drug, protein, nucleic acid, (e.g. mRNA),carbohydrate, or lipid; consciousness of the subject, cognitivefunctions, self-administration paradigms, reward-stimulus paradigms,electrophysiological functions, and memory.

The invention provides a variety of methods for identification,diagnosis, and treatment of neuropathologies, neurotoxicities, tumors,and brain and spinal cord injuries using electrodes withmicrovoltammetry. These methods comprise comparing Broderick probemicrovoltammograms from at least two different tissues. One thesetissues is generally a reference tissue or control. The other is tissueis that being assayed. Preferably, the reference tissue corresponds tothe assay tissue with respect to, for example, tissue type, anatomicallocation, and/or stage of development.

One embodiment of the instant invention utilizes the sensors foridentifying particular biomolecules, where the sensors may or may not bepre-treated with the biomolecules. Identifying the presence of specificbiomolecules may result in the diagnosis and treatment of diseases,conditions, or disorders, as well as, of any addiction. Biomoleculesinclude but are not limited to, pharmaceutical compounds, pharmaceuticalcompounds specific for neurodegenerative or neuropsychiatric diseases,disorders, and conditions, neurotransmitters, neuromodulators, hormones,surfactants, soaps, detergents, pramipexole, topiramate, clozapine,dopamine, serotonin, norepinephrine, acetylcholine, adenosine, estrogen,vitamins, vitamin A, vitamin E, brain lipids, phosphotidylethanolamine,tallow, sodium lauryl sulfate, N-acetyl-aspartate, choline, lactate,uric acid, stabilizing proteins, amyloid proteins, ascorbic acid,γ-aminobutyric acid, glutamate, neurotensin, somatostatin, dynorphin,homovanillic acid, nucleic acids, tryptophan, tyrosine, nitrous oxide,nitric oxide, or combinations thereof. Changes in the presence orabsence of biomolecules compared to normal conditions assist in thedetermination of, for example, disease, cancer, malignant or benign,tumor growth, drug addiction, or necrosis. Biomolecules are used asmarkers to diagnose and treat diseases, conditions, or disorders. Somebiomolecules are particularly good as markers for certain diseases,conditions, or disorders. For example, uric acid, N-acetyl-aspartate,choline, and lactate are effective markers for brain tumors. Effectivemarkers for epilepsy include, but are not limited to,N-acetyl-aspartate, choline, creatinine, dopamine, seratonin, glutamate,and γ-aminobutyric acid. Dopamine is also a useful marker forParkinson's disease. Whereas for schizophrenia and drug addictions, suchas for example cocaine addiction, dopamine and serotonin biomoleculesare useful.

In some embodiments of the invention, the comparison is performedbetween microvoltammograms taken from the same tissue at differenttimes. In some embodiments, the microvoltammograms compared are takenfrom the same tissue before and after exposure to a material such as adrug. In some embodiments, a tissue suspected of being diseased iscompared with healthy tissue.

Such comparisons may make it possible to diagnose and/or treat a widevariety of diseases or conditions that are associated with abnormalneurotransmitter levels. The invention provides methods comprisingexposing at least a cell to a diagnostic challenge or therapeutictreatment, contacting said cell with a Broderick porbe, applying apotential to said Broderick probe; and generating a Broderick probemicrovoltammogram. A diagnostic challenge may be designed to elicit adifferential response from cells of interest, e.g. diseased cells, fromother cells, e.g. healthy cells. A therapeutic treatment may ortherapeutic treatment may be known or intended to cure or ameliorate adisease condition. Alternatively, a treatment may be assessed for itscapacity to serve as a diagnostic indicator or therapeutic treatment. Adiagnostic challenge or a therapeutic treatment may comprise exposingthe cell(s) to a material such as a small molecule drug or drugcandidate, a defined electrochemical environment (e.g. application of apotential to the cell(s)), exposure to an isotopic or nonisotopic label,activation or repression of a preselected gene, or combinations thereof.

Disorders of basal ganglia, such as athetoid, dystonic diseases, andcancer may be studied with the Broderick probe. An example of anathetoid, dystonic disease is Lesch Nyhan Syndrome (LNS). This recentlyrecognized disease is characterized by severe athetoid and dystonicmovements, self-mutilation, and repetitive oral stereotypies. Patientssuffering from LNS may have to have their teeth removed to avoid oralstereotypies that cause the patient to devour lips, tongues or fingers.The stereotypies involve DA and 5-HT (Allen S M et al., 1999, Behav.Pharmacol. 10:467-474) and high levels of UA (Patten J, 1980,Neurological Differential Diagnosis, pp. 127-128). Other athetoid anddystonic diseases, such as autism, spinal cord injury, schizophrenia,epilepsy and Parkinson's, are amenable for study with these miniaturesensors, even intraoperatively, insofar as epilepsy and Parkinson's areconcerned. Several reports indicate that various cancers are alsoamenable for study with these miniature sensors (Broderick P A, 1989,U.S. Pat. No. 4,883,057; Broderick P A, 1995, U.S. Pat. No. 5,433,710;Broderick P A, 1997, EP 0487647 B1; Broderick P A, 1999, U.S. Pat. No.5,938,903; Broderick P A, 1999, Hong Kong, HK #1007350; Broderick P A,1988, Neurosci. Lett. 95:275-280; Broderick P A, 1989, Brain Res.495:115-121; Broderick P A, 1990, Electroanalysis 2:241-251; Broderick PA, 2000, Epilepsia 41(Suppl.):91; Broderick P A et al., 2000, Brain Res.878:49-63).

Much of the difficulty in determining the importance of the alterationsin relative concentrations of neurotransmitters and their relationshipto epileptogenesis in temporal lobe epilepsy relates to the variabilityin both the etiology of epilepsy and the location of the epileptogeniczone in epilepsy patients. Few studies have analyzed monoamineconcentrations in human epileptic tissue. Those that have studiedresected temporal lobe tissue have not distinguished between neocorticaltemporal lobe epilepsy and mesial temporal lobe epilepsy patients.Neocortical temporal lobe tissue that was part of the ictal onset zoneas verified by intracranial EEG recordings of seizures in patients withneocortical temporal lobe epilepsy was examined according to theinvention. As previously described (Doyle W K et al., 1997, Epilepsy: AComprehensive Textbook, pp. 1807-1815), the anterior temporal neocortexin patients with mesial temporal lobe epilepsy is routinely removed atour center to gain access to the mesial temporal structures, providingneocortical tissue controls for our study. While patients with mesialtemporal lobe epilepsy may have coexisting neocortical abnormalitieslike cortical dysplasia (CD), none of the mesial temporal lobe epilepsypatients included in this study had pathologically confirmed CD.Secondary changes such as mild diffuse gliosis were found in thetemporal neocortex of mesial temporal lobe epilepsy patients, but wehypothesized that the normal neurochemical profile may still bepreserved compared to the actively seizing neocortical tissue analyzedin our neocortical temporal lobe epilepsy patients.

The invention provides devices and methods for diagnosing temporal lobeepilepsy comprising generating a temporally resolved Broderick probemicrovoltammogram of a temporal lobe tissue of a subject; and comparingsaid microvoltammogram to at least one reference Broderick probemicrovoltammogram; wherein said reference is a Broderick probemicrovoltammogram of the corresponding temporal lobe tissue of anotherindividual. In some embodiments of the invention, the subject'smicrovoltammogram is compared with one or more reference or controlmicrovoltammograms from a healthy individual, an individual havingmesial temporal lobe epilepsy, an individual having neocortical temporallobe epilepsy, or combinations thereof.

The invention provides diagnostic devices and methods for brain cancer.In some embodiments the methods comprise: generating a temporallyresolved Broderick probe microvoltammetric profile of cancerous cells ortissue; determining from said profile the presence and concentration ofat least two markers or biomolecules comprising pharmaceuticalcompounds, neurotransmitters, neuromodulators, hormones, surfactants,soaps, detergents, dopamine, serotonin, norepinephrine, acetylcholine,adenosine, estrogen, vitamins, vitamin A, vitamin E, brain lipids,phosphotidylethanolamine, tallow, sodium lauryl sulfate,N-acetyl-aspartate, choline, lactate, uric acid, stabilizing proteins,amyloid proteins, ascorbic acid, γ-aminobutyric acid, glutamate,neurotensin, somatostatin, dynorphin, homovanillic acid, nucleic acids,tryptophan, tyrosine, nitrous oxide, nitric oxide, or combinationsthereof, and comparing said marker or biomolecule concentrations tospecific threshold values of each of the markers to determine thepresence of statistically significant concentration differences,preferably P<0.05; wherein said threshold values are derived fromBroderick probe microvoltammetric profile(s) of healthy cells or tissueand said step of comparing said markers distinguishes whether thecancerous cells are present in gray matter or white matter. In otherembodiments, the diagnostic methods comprise generating a temporallyresolved Broderick probe microvoltammetric profile of a tissue having orat risk of having a tumor; comparing said microvoltammogram to at leastone reference Broderick probe microvoltammogram; wherein said referenceis a Broderick probe microvoltammogram of corresponding tissue of ahealthy individual, cultured cells thereof, corresponding tissue of a anindividual having a tumor, cultured cells thereof, or combinationsthereof.

The invention also provides diagnostic devices and methods for brain orspinal cord injury. In some embodiments of the invention these methodscomprise: generating a temporally resolved Broderick probemicrovoltammogram of a tissue of a mammal having or being at risk ofdeveloping a brain or spinal cord injury; simultaneously monitoringmovement of said mammal; and comparing said microvoltammogram andmovement behavior to a reference or control microvoltammogram ofcorresponding tissue of a healthy tissue and reference movement behaviorof a healthy individual. In addition, the invention provides methods fordetecting a site of nerve damage or blockage. These methods may comprisegenerating a temporally resolved Broderick probe microvoltammogram of atissue of said mammal; simultaneously monitoring movement of saidmammal; and comparing said microvoltammogram and movement behavior to areference microvoltammogram of corresponding tissue of a healthy tissueand reference movement behavior of a healthy individual, where themicrovoltammogram uses the inventive sensor.

The invention provides devices and methods for treating temporal lobeepilepsy comprising generating a temporally resolved Broderick probemicrovoltammogram of a temporal lobe tissue of a subject having or atrisk of developing a temporal lobe epilepsy; comparing saidmicrovoltammogram to at least one reference Broderick probemicrovoltammogram; determining the type and extent of temporal loberesection necessary to achieve a substantially seizure free outcome; andresecting the subject's temporal lobe accordingly. In some embodimentsof the invention, the subject's microvoltammogram is compared with oneor more reference microvoltammograms from a healthy individual, anindividual having mesial temporal lobe epilepsy, an individual havingneocortical temporal lobe epilepsy, or combinations thereof.

Brain or spinal cord injuries as well as nerve damage or blockagetreatment may include generating Broderick probe microvoltammogramsduring therapy, i.e. while a pharmacological therapy or kinesitherapy isbeing administered. Broderick probe microvoltammograms may be acquiredcontinuously during therapy or at intervals. Likewise, cancer treatmentsmay be adapted to include Broderick probe microvoltammetry duringtherapy. By generating Broderick probe microvoltammograms, it may bepossible to monitor tumor size.

The invention contemplates the use of microvoltammetry to assess theneurotoxicity of any material. In some embodiments of the invention,Broderick probe microvoltammograms are acquired from a neural cell ortissue in the presence and absence of the subject material. Materialsthat may be tested include controlled substances (e.g. opiates,stimulants, depressants, hallucinogens), anti-depressants, anti-epilepsydrugs, and other psychopharmacological substances.

The term “controlled substances” refers to all substances listed in 21C.F.R. §1308 even where those referenced only as exceptions. It furtherincludes all salts, geometric and stereoisomers, and derivatives ofsubstances listed therein.

Opiates include, inter alia, alfentanil, alphaprodine, anileridine,apomorphine, bezitramide, carfentanil, cocaine, codeine,4-cyano-2-dimethylamino-4, 4-diphenyl butane,4-cyano-1-methyl-4-phenylpip- eridine pethidine-intermediate-B,dextropropoxyphene, dextrorphan, dihydrocodeine, dihydroetorphine,diphenoxylate, 1-gdiphenylpropane-carbo- xylic acid pethidine(meperidine), ecgonine, ethyl-4-phenylpiperidine-4-ca-rboxylatepethidine-intermediate-C, ethylmorphine, etorphine hydrochloride,fentanyl, hydrocodone, hydromorphone, isomethadone,levo-alphacetylmethadol, levomethorphan, levorphanol, metazocine,methadone, methadone-intermediate,2-methyl-3-morpholino-1,1-methyl-4-phenylpiperidine-4-carboxylic acid,metopon, morphine, moramide-intermediate, nalbuphine, nalmefene,naloxone, naltrexone, opium, oxycodone, oxymorphone,pethidine-intermediate-A, phenanthrene alkaloidsphenazocine, piminodine,racemethorphan, racemorphan, remifentanil, sufentanil, thebaine, andthebaine-derived butorphanol.

Stimulants include substances having a stimulant effect on the centralnervous system such as, inter alia, amphetamine, methamphetamine,phenmetrazine, methylphenidate, and salts, isomers, and salts of isomersthereof.

Depressants include substances having a depressant effect on the centralnervous system such as, inter alia, amobarbital, glutethimide,pentobarbital, phencyclidine, and secobarbital

Hallucinogens include, inter alia, nabilone.

Anti-depression drugs include, inter alia, citalopram, fluvoxamine,paroxetine, fluoxetine, sertraline, amitriptyline, desipramine,nortriptyline, venlafaxine, pheneizine, tranylcypromine, mirtazepine,nefazodone, trazodone, and bupropion.

Anti-epilepsy drugs include, inter alia, carbamazepine, clorazepate,clopazine, ethosuximide, felbamate, gabapentin, lamotrigine,phenobarbital, phenytoin, primidone, topiramate, and valproic acid.

The neurological side effects including neurotoxicity of anypharmaceutical may be assayed according to the methods of the invention.Neurotoxicity of other substances such as minerals, ions, metals (e.g.heavy metals such as mercury and lead), caffeine, ethanol, nicotine,cannabinoids proteins, lipids, nucleic acids, carbohydrates,glycolipids, and lipoproteins may also be assessed using methods of theinvention.

The contents of all patents, patent applications, published PCTapplications and articles, books, references, reference manuals andabstracts cited herein are hereby incorporated by reference in theirentirety to more fully describe the state of the art to which theinvention pertains.

As various changes may be made in the above-described subject matterwithout departing from the scope and spirit of the present invention, itis intended that all subject matter contained in the above description,or defined in the appended claims, be interpreted as descriptive andillustrative of the present invention. Many modifications and variationsof the present invention are possible in light of the above teachings.

EXAMPLES

The Examples herein are meant to exemplify the various aspects ofcarrying out the invention and are not intended to limit the scope ofthe invention in any way. The Examples do not include detaileddescriptions for conventional methods employed that are well known tothose skilled in the art and are described in numerous publications.Other embodiments will be apparent to one of ordinary skill in the artand do not depart from the scope of the invention. All referencesdescribed herein are expressly incorporated in toto by reference.

Example 1

BRODERICK PROBE® sensors may have different formulations, e.g.,cerebrosides and oleic acid; however, for these studies, the lauratesensor was used because it reaches steady state quickly. Although thesesensors can be manufactured at any size or any length, for thesestudies, the laurate sensors comprising an indicator and construction.The construction portion was made of a polytetrafluoroethylene (PTFE;Teflon®) coated stainless steel wire having a diameter of 150 μm(Medwire, Mount Vernon, N.Y.). The coating was gently pulled 500 μm overthe stainless steel tip to form a well. The well, or indicator portion,was packed with a graphite paste modified with (99+%) lauric acid. Thegraphite/lauric acid (Ultra Carbon, Bay City Mich.) (Sigma, St. Louis,Mo.) was admixed with mineral oil (a.k.a. Nujol) containing DL-alphatocopherol as a stabilizer (Plough Inc., Memphis, Tenn.).

The reference microelectrode was constructed by electrochemicallycoating silver wire (Medwire, Mount Vernon, N.Y.) with chloride ions byapplying a 2mA current to the silver wire in a 1M NaCl solution forone-half hour. The Ag/Ag/Cl reference should not differ from anycommercial Ag/Ag/Cl reference microelectrode by more than 2.5 mV.Reference microelectrodes should be stored in 50 microliters ofphysiological saline.

The auxiliary microelectrode was a simple construction of stainlesssteel, covered with a polytetrafluoroethylene (PTFE; Teflon®) with thestainless steel removed at the tip (diameter=150 um) (Broderick, P. A.,1995, U.S. Pat. No. 5,433,710; Broderick, P. A., 1999, U.S. Pat. No.5,938,903.; Broderick P A and Pacia, S V, U.S. Pat. No. Ser. No.10/118,571, 2006).

Combustion analyses and gas chromatographic analyses were performed on 5mg of the mixture to determine the graphite content and the lauric acidcontent, respectively. Each sensor used approximately 1 mg of themixture.

Example 2 Human Epilepsy

Fourteen patients who had temporal lobectomies for intractable seizureswere studied. Patients underwent intraoperative surgery within the sametime period and were studied in order of time, within the same timeperiod. Patients were classified as having mesial temporal lobe epilepsyif pathologic examination of the resected temporal lobe revealed severehippocampal neuronal loss and gliosis and if examination of theneocortex revealed no other etiology for the patient's epilepsy. Ninepatients were classified as mesial temporal lobe epilepsy based on thesefeatures. Five patients were classified as having neocortical temporallobe epilepsy based on the lack of hippocampal atrophy on magneticresonance imaging (MRI) and demonstration of seizure onset in temporalneocortex during chronic intracranial EEG study with lateral temporalsubdural grid electrodes and multiple baso-mesial temporal subduralstrip electrodes.

FIG. 1 shows a schematic diagram of a Broderick Probe electrode. Theelectrode manufacturing process has been published in detail elsewhere(Broderick P A, 1993, Pharmacol. Biochem. Behav. 46:973-984; Broderick PA, 2002, Handbook of Neurotoxicology, Vol. 2, Chapter 13; Broderick P Aet al., 2000, Brain Res. 878:48-63). Studies revealed no promotion ofbacterial growth on the electrodes with and without gamma irradiation(Sterigenics, N.C.).

The in vivo microvoltammetric measurement was made through theapplication of a potential (in mV) between the indicator (working)electrode and a Ag/AgCl reference electrode. Current is formed at theindicator electrode that corresponds to the separate electroactivespecies for dopamine, serotonin, norepinephrine, and ascorbic acid. Theresultant electrochemical measurements are called Faradaic because theamount of the oxidative/reductive species detected at the electrodesurface is calculated by Faraday's Law, which shows that a directproportionality exists between the charge and the mass of a chemical.The proportionality between charge and mass is described by the Cottrellequation.

Potentials were applied to the working electrode with respect to theAg/AgCl electrode by a CV37 detector-potentiostat, electricallyconnected to a Minigard Surge Suppressor which is then connected to anisolated electric ground. Each neurotransmitter and neuromodulator wasdetected within seconds of release at a scan rate of IOmV/s (see FIGS.3A&B). In vivo microvoltammetric signatures for neurotransmitters andneuromodulators were determined by experimentally established oxidationpotentials. Oxidation potentials were delineated in millivolts.Additionally, lauric acid electrodes may be employed for intraoperativerecordings due to their extremely rapid equilibration time (Broderick PA et al., 1999, Epilepsia 40(supp 17):78-79).

Resected temporal lobe tissue from 14 epilepsy patients was examined ina medium of Ringer's Lactate Buffer solution. Samples were taken fromthe antero-lateral temporal neocortex. In a faradaic chamber, areference and an auxiliary electrode were placed in contact with thesurface of the specimen and a stearate indicator electrodestereotaxically was inserted dorsoventrally, about 2 mm into the braintissue, in situ. With the triple electrode assembly in place, potentialswere applied and scanned at a rate of 10 mV/sec from an initial voltageof −0.2 V up to a voltage of +0.9 V. The electrochemical signals wererecorded on a strip chart recorder.

Table 1 lists the concentrations of norepinephrine, serotonin, anddopamine found in the lateral temporal neocortex (Band of Baillarger) ofthe nine mesial temporal lobe epilepsy and five neocortical temporallobe epilepsy patients. Representative Broderick probe voltammogramsfrom patients 8 and 14 are shown in FIG. 4. Four of five neocorticaltemporal lobe epilepsy patients had no detectable norepinephrine intemporal neocortex while norepinephrine was present in temporalneocortex of eight of nine mesial temporal lobe epilepsy patients (ChiSquare, p<0.01). The mean norepinephrine concentration was 21.1 nM±5.8nM. Statistical significance was analyzed by the median according to theMann-Whitney Rank Sum, (p<0.065) for the mesial temporal lobe epilepsygroup. In contrast dopamine was detected in the temporal neocortex ofthree of five neocortical temporal lobe epilepsy patients but in onlyone of the mesial temporal lobe epilepsy patients (Chi Square, p<0.05).The mean dopamine concentration for the neocortical temporal lobeepilepsy group was 16.7 nM±7.6 nM. Individual dopamine concentration foreach neocortical temporal lobe epilepsy patient was compared withdopamine concentration from each mesial temporal lobe epilepsy patient.Dopamine was significantly greater in neocortical temporal lobe epilepsyvs. mesial temporal lobe epilepsy (Mann-Whitney Rank Sum, p<0.027).TABLE 1 Monoamine Concentrations in the Neocortex (Band of Baillarger)in MTLE and NTLE Patients. Patient # MTLE Patient # NTLE 1 NE 5-HT 10 DA5-HT 48.3 1.5 17.8 2.0 2 NE 5-HT 11 DA 5-HT 8.6 1.7 29.2 2.1 3 NE 5-HT12 AA 5-HT 5.6 1.3 0.11 4.9 4 NE 5-HT 13 NE 5-HT 4.3 1.3 44.8 1.7 5 NE5-HT 14 DA 5-HT 17.2 1.9 3.0 2.2 6 DA 5-HT 17.2 2.0 7 NE 5-HT 25.8 3.5 8NE 5-HT 8.5 3.2 9 NE 5-HT 11.6 3.6

Pathology for patients 1 through 9 is hippocampal disease; pathology forpatients 10 through 14 is neocortical disease. Values for monoamines[Norepinephrine (NE), Serotonin (5-HT), Dopamine (DA)] are calibrated innanomolar (nM) concentrations. Values for Ascorbic Acid (AA) werecalibrated in micromolar (μM) concentrations. Neurochemical signaturesfor mesial temporal lobe epilepsy patients consisted of the presence ofnorepineplrrine and 5HT and the absence of dopamine in each neocorticalspecimen with the exception of patient #6, who exhibited dopamine andnot norepinephrine. Neurochemical signatures for neocortical temporallobe epilepsy patients consisted of the presence of dopamine, serotoninand ascorbic acid and the absence of norepinephrine in each neocorticalspecimen with the exception of patient, #14 who exhibited norepinephrineand not dopamine.

Serotonin was detected in the temporal neocortex of all 14 patients,with a mean concentration of 2.2 nM±0.32 for the mesial temporal lobeepilepsy patients and 2.58 nM±0.59 for the neocortical temporal lobeepilepsy group. Ascorbic acid was found in the temporal neocortex of oneof the neocortical temporal lobe epilepsy patients at a concentration of0.1 .mu.M, but in none of the mesial temporal lobe epilepsy patients.

The most striking finding of this study was the marked norepinephrinedepletion seen in the temporal neocortex of neocortical temporal lobeepilepsy patients compared with that of the mesial temporal lobeepilepsy patients. In experimental models, norepinephrine maydifferentially enhance or inhibit GABAergic cells (Kawaguchi Y et al.,1998, J Neurosci 18:6963-6976). However, in numerous experimental modelsof epilepsy, norepinephrine depletion has been shown to enhance thefrequency, intensity, and spread of seizures (Browning R A et al., 1989,J Pharmacol Exp Ther 249:229-35; Ferrendelli J A et al., 1986, Adv.Neurol. 44:393-400). A consistent depletion in neocorticalnorepinephrine has not been previously demonstrated in human epilepsy.One study by Goldstein et al. (Goldstein D S et al., 1988, J Neurochem50:225229), using intraoperative electrocorticography, showed increasedconcentrations of norepinephrine in spiking cortex compared withnonspiking cortex. This increased concentration was hypothesized to be acompensatory, inhibitory role of norepinephrine. However, this studyused whole tissue homogenates of large cortical regions and it isdifficult to compare these data to the restricted neocortical samplesexamined in this Example. Subtypes of temporal lobe epilepsy were alsonot considered. In another study of 20 patients with TLE by Pintor andcolleagues, no difference in norepinephrine concentrations were foundbetween spiking and nonspiking regions (Pintor M et al., 1990, Synapse5:152-156).

These results suggest that the Band of Baillarger is white matter. TABLE2 Statistical Results. 1) Results showed that the neurotransmitter DA DAin temporal neocortex of NTLE patients was NTLE > MTLE significantlygreater that in MTLE patients: Chi-square -p < 0.05 for presence vs.absence ofDA; Mann-Whitney Rank Sum - p = 0.027 (p < 0.01) forconcentration of DA in NTLE to be significantly greater than that ofMTLE 2) 5-HT: Chi-square (not applicable); Mann-Whitney 5-HT Rank Sum -p = 0.317. Not Significant (N.S.) N.S. 3) NE concentrations in temporalneocortex were NE significantly greater in MTLE than in NTLE MTLE > NTLEpatients: Chi-square -p < 0.01; Mann- Whitney Rank Sum - p = 0.065 (p <0.01) 4) AA: Chi-square - p < 0.2; Mann-Whitney Rank AA Sum - p = 0.894.NTLE > MTLE Trend: Chi square

The most striking finding of this study was the marked norepinephrinedepletion seen in the temporal neocortex of our neocortical temporallobe epilepsy patients compared with that of the mesial temporal lobeepilepsy patients. In experimental models, norepinephrine maydifferentially enhance or inhibit GABAergic cells (Kawaguchi Y et al.,1998, J Neurosci 18:6963-6976). However, in numerous experimental modelsof epilepsy, norepinephrine depletion has been shown to enhance thefrequency, intensity and spread of seizures (Browning R A et al., 1989,J Pharmacol Exp Ther 249:229-35; Ferrendelli J A et al., 1986, Adv.Neurol. 44:393-400). To our knowledge, a consistent depletion inneocortical norepinephrine has not been previously demonstrated in humanepilepsy. One study by Goldstein et al. (Goldstein D S et al., 1988, JNeurochem 50:225229), using intraoperative electrocorticography, showedincreased concentrations of norepinephrine in spiking cortex comparedwith nonspiking cortex. This increased concentration was hypothesized tobe a compensatory, inhibitory role of norepinephrine. However, thisstudy used whole tissue homogenates of large cortical regions and it isdifficult to compare these data to the restricted neocortical samples inour study. Subtypes of temporal lobe epilepsy were also not considered.In another study of 20 patients with TLE by Pintor and colleagues, nodifference in norepinephrine concentrations were found between spikingand nonspiking regions (Pintor M et al., 1990, Synapse 5:152-156).

Example 3 Human Epilepsy

Significant differences in the monoamine signatures from the hippocampalsubparcellations in patients with MTLE and NTLE have been observed. Thealveus (hippocampal white matter) contains both efferent fibers fromhippocampus that form the fornix and afferent pathways connectingentorhinal cortex and the CA1 region of the hippocampus. Theneurochemistry of the alveus in patients with MTLE and NTLE was studiedto determine whether similar neurotransmitter alterations exist.

Microvoltammetry with Broderick probe stearic acid electrodes was usedto detect norepinephrine (NE), dopamine (DA), ascorbic acid (AA), andserotonin (5-HT) in resected temporal lobes of 9 MTLE and 4 NTLEpatients with temporal lobe epilepsy. Neurotransmitters were detected inseparate signals within the same recording within seconds in alveus byexperimentally derived oxidative potentials, determined in vitro inRingers Lactate or PO₄ buffer. Ag/AgCl reference and stainless steelauxiliary micro electrodes were placed in each specimen 4-6 mm fromindicator electrodes (patented)(manufactured on site). Methods arepublished (Broderick P A, 1989, Brain Res. 495:115-121; Broderick P A,1988, Neurosci. Lett. 95:275-280; Broderick P A et al., 2000, Brain Res.878:48-63; Pacia S V, 2001, Brain Res. 899(1-2):106-11). All signalswere analyzed for the presence of diffusion peaks in early oxidativespecies (indicating white matter).

All of the NTLE patients had significant DA in the alveus while only oneMTLE patient had detectable DA (p<0.01, Mann-Whitney Rank Sum Test).Eight of the nine MTLE patients had significant NE in alveus while noneof the concentrations were significantly higher in NTLE specimens(p<0.01, Mann-Whitney Rank Sum Test).

The neurochemical profiles in alveus of patients with MTLE and NTLEreveal neurotransmitter alterations similar to those alterations seen inthe hippocampal pyramidal cell layer of these patients, a region withdirect afferent and efferent connections through the alveus.

Example 4 Distinguishing White and Gray Matter

Voltammetric signals were analyzed from electrodes in resected temporallobes to determine whether gray and white matter structures could bereliably distinguished.

Microvoltammetry with Broderick Probe stearic acid electrodes was usedto detect norepinephrine (NE), dopamine (DA), ascorbic acid (AA), andserotonin (5-HT) in 40 gray matter structures and 37 white matterstructures in resected temporal lobes of a total of 14 patients withtemporal lobe epilepsy. Neurotransmitters were detected in separatesignals within the same recording within seconds in 3 gray matter(temporal neocortex, hippocampal pyramidal, and dentate gyrus granularlayer) and 3 white matter structures (temporal stem, subiculum, andalveus), by experimentally derived oxidative potentials, determined invitro in Ringers Lactate or PO₄ buffer. Ag/AgCl reference and stainlesssteel auxiliary electrodes were placed in each specimen 4-6 mm fromindicator electrodes (patented)(manufactured on site). Methods arepublished (Broderick P A, 1989, Brain Res. 495:115-121; Broderick P A,1988, Neurosci. Lett. 95:275-280; Broderick P A et al., 2000, Brain Res.878:48-63; Pacia S V, 2001, Brain Res. 899(1-2):106-11). All signalswere analyzed for the presence of diffusion peaks in early oxidativespecies (indicating white matter).

FIG. 5 shows representative examples of distinctive signals recordedfrom gray matter (A) and white matter (B). Criteria for distinguishingelectrochemical signals for gray versus white matter consist of distinctdifferences in the catecholamine (i.e. dopamine, norepinephrine) peaks,peaks that are called the “early oxidative species”. They are asfollows:

(A) Gray matter signals are large in amplitude denoting highconcentrations, whereas white matter signals are small in amplitudedenoting low concentrations of neurotransmitters. Moreover, white mattersignals are defined electrochemically as “broad diffusion waveforms”.

Criteria for denoting distinct differences in the indoleamine peaks,e.g. serotonin, peaks that are exhibited later than the catecholaminesin the oxidative sweep pathway, consist of the following:

(B) Gray matter signals for serotonin are generally lower than those inwhite matter. White matter signals exhibit a sharp adsorptive waveform.

Of 40 temporal lobe gray matter structures sampled, 39 displayed signalsconsistent with gray matter and only 1 revealed signals consistent withwhite matter. Of 37 white matter structures sampled, 30 exhibitedsignals consistent with white matter. Additionally, white matter tendedto have substantially lower concentrations of catecholamines and 5-HT aswell as lower ratios of catecholamines to 5-HT. In some preferedembodiments of the invention, the first peak, i.e. catecholamine, forwhite matter is about two-fold less than the same peak for white matter.In some prefered embodiments, the second peak, i.e. serotonin, for whitematter is about two-fold more than the same peak for white matter.

Gray matter has an inherent neuroanatomic difference from white matterand this difference may explain specific waveforms for gray matterversus white matter signals. In gray matter, calcium ions course intofibers through calcium channels which gate neurotransmitters. Calciumchannels are absent in white matter and gating of neurotransmittersoccurs through sodium channels. In situ microvoltammetry with electrodesreliably distinguishes temporal lobe gray matter from white matter, bothin the neocortex and in other neuroanatomic substrates such as specifichippocampal parcellations that are comprised of white matter versus graymatter. These results have important implications for in vivo and/orintraoperative neurochemical analysis of human epilepsy. These findingsshould enable more precise intraoperative neuroanatomic localization.

Furthermore, these findings have important implications fordistinguishing and locating tumors/neoplasms and the like,intraoperatively and otherwise especially when these tumors whether theybe associated with epilepsy or not, infiltrate other tissues. To date,magnetic resonance imaging (MRI) is unable to detect tumors infiltratedto other tissues.

Example 5 Distinguishing White and Gray Matter

This example is directed to elucidating significant differences in thelevels of catecholamine neurotransmitters, dopamine (DA), norepinephrine(NE) and indolamine neurotransmitter, serotonin (5-HT) in the alveus andtemporal stem of 14 intractable epileptic patients, including 9 MesialTemporal Lobe Epilepsy (MTLE) and 5 Neocortical (lateral) Temporal LobeEpilepsy (NTLE) patients who underwent surgery. DA, NE, and 5-HT weredetected separately according to their experimentally establishedoxidative potentials. This detection was achieved with in vivoelectrochemistry, which depended on a semi-differentialmicrovoltammetry-based system of electrodes, consisting of a miniaturecarbon sensor (BRODERICK PROBE®), Ag/AgCl reference electrode, andauxiliary micro electrode. Methods are published by Broderick (BroderickP A, 1989, Brain Res. 495:115-121; Broderick P A, 1988, Neurosci. Lett.95:275-280). Relative to NTLE specimens, MTLE specimens exhibited areduced level of DA and higher level of NE in alveus and temporal stem.Although, 5-HT was common to all specimens, NTLE specimens showed asignificantly higher level in the alveus. The data indicate that MTLEand NTLE are characterized by distinct neuronal microenvironment andthat epilepsy-associated gliosis could affect extraneuronal monoamine.

Classically, glial cells have been considered to comprise a passiveframework that supports, nourishes, and insulates neurons. They have notbeen thought of as active factors in the onset and progression of braindisease such as epilepsy. Epilepsy is frequently defined as a neuronaldisease marked by spontaneous recurring seizures accompanied bysignificant biochemical imbalances. Recent studies show that glialcells—the major component of alveus and temporal stem—are activelyinvolved in regulating extraneuronal ions (Bordey A et al., 1998,Epilepsy Res. 32:286-303; Walz W, 1989, Prog. Neurobiol. 33: 309-333)neurotransmitters (Kimelberg H K et al., 1993, Astrocytes, Pharmacologyand Function, pp. 193-228) that are involved in the propagation ofepilepsy. Electron microscopic sections also show that astrocytesencapsulate neuronal cell bodies and reach closely into the vicinity ofsynapses. Such anatomical intimacy gives astrocytes access to regulatethe neuronal microenvironment.

Numerous studies clearly indicate the relevance of monoamines and theirregulation by glial cells towards the propagation of epilepsy. Theinstant example illustrates how electrodes may be used to delineate thelevels of catecholamines and serotonin in the hippocampal andneocortical white layers of epileptic tissue. Specifically, the systemof electrodes utilized herein made it possible to delineate the levelsof DA, NE, and 5-HT in the alveus and temporal stem. Consequently, itwas possible to assess the impact of epileptic seizure and itsconsequences on the regulation of extraneuronal catecholamines andserotonin in MTLE and NTLE tissues by comparing neurotransmitter levelsbefore and during a seizure.

Hippocampal and temporal cortex resected from fourteen intractableepilepsy patients was immersed in Ringer's Lactate Buffer Solution. Aminiature carbon sensor (BRODERICK PROBE®) was inserted dorsoventrally(1 mm or 2 mm) into alveus and temporal stem, while a Ag/AgCl referenceand auxiliary electrodes were placed in contact with the surface of thespecimen. This system of electrodes has been proven capable of detectingcatecholamine DA, NE and indolamine 5-HT neurotransmitters and theirmetabolites. Methods are published (Broderick P A, 1989, Brain Res.495:115-121; Broderick P A, 1988, Neurosci. Lett. 95:275-280).Potentials were applied and scanned at a rate of 10 mV/sec from −0.2V to+0.9V across the reference and indicator electrodes via a CV-37Electrochemical Detector (BAS, West Lafayette, Ind.). Each of the 4monoamines was detected separately at its characteristic oxidativepotential, which was experimentally determined (Broderick P A, 1989,Brain Res. 495:115-121; Broderick P A, 1988, Neurosci. Lett.95:275-280). The resulting electrochemical signals were recorded on aFisher Recordall.RTM. Series 5000 (Houston instruments, Houston, Tex.).The data were statistically analyzed using Chi-square Test, and MannWhitney Rank Sum Test.

In the alveus, there was a significant decrease in the concentrations ofDA in MTLE specimens relative to NTLE specimens (p=0.011; Mann-WhitneyRank Sum Test), and a significant presence of NE in NTLE specimensrelative to MTLE (p<0.01; Chi-square). There was a significant depletionin the concentration of NE in NTLE alveus specimens relative to MTLE(p=0.031; Mann-Whitney Rank Sum Test), and a significant occurrence ofNE in MTLE relative to MTLE specimens (p<0.001; Chi-square). Althoughthe concentrations of 5-HT are significantly higher in NTLE alveusspecimens (p=0.045; Mann-Whitney Rank Sum Test). The actual recordingfor Patient #7 is shown in FIG. 4A. Patient #7 was diagnosed as havingmesial temporal lobe epilepsy.

There was depletion of NE in the NTLE temporal stem specimens relativeto MTLE temporal stem specimen [p=0.082]. Chi-square test showed asignificant presence of NE in MTLE relative to NTLE specimens (p<0.05).There was no statistical difference in the concentration and occurrenceof 5-HT in temporal stem (white matter) MTLE and NTLE specimens. Theactual recording for Patient #7 is shown in FIG. 4B.

The results indicate a notable dysfunction in the extraneuronalregulation of (a) DA, NE, and 5-HT in the alveus of MTLE and NTLEpatients, and (b) DA and NE in temporal stem of NTLE versus MTLE. Theseextraneuronal trends extend previous findings that “subtypes of epilepsyare associated with distinct neuronal biochemistry in human neocortexand hippocampus of MTLE and NTLE patients” (Broderick P A et al., 2000,Brain Res. 878:48-63). These differences are extended to white matterhippocampal subparcellations such as alveus and neocortical white mattersuch as temporal stem.

The significant depletion of DA in the alveus of MTLE specimens may beinterpreted as (i) the outcome of increased compensatory DA metabolismin the neuronal microenvironment either by dopamine-.beta.-hydroxylaseto NE; or possibly by monoamine oxidase to DOPAC; or still bytryosinase, prostaglandin H synthase and/or xanthine oxidase to thetoxic DA quinone; (ii) indication of dopaminergic neuronal death, anevidence of the cytotoxic effect of DA quinone. This explanationconforms with the model of cytotoxic and genotoxic potential of DA viaDA quinone, as advanced by (Stokes A H et al., 1999, J. Neurosci. Res.55:659-665).

Electrodes with microvoltammetry as described in the present inventionmay be utilized to study movement disorders whether they originate inthe brain or the spinal cord. This is because this is the firsttechnology that can detect neurotransmitters at the same time thatmovement occurs. Thus, for spinal cord injury, e.g., Broderick probesare implanted or inserted into neurons and/or interneurons of muscle andganglia either in situ or in vivo, dysfunction between neurotransmitterand movement is directly detected for appropriate therapeuticinterventions. The following examples show how normal behavior, which isrhythmic, needs to be rhythmic with the neurotransmitter, serotonin fornormal functioning of brain and spinal cord. This example has beendescribed previously in this application. When injury occurs, irregularsynchrony between neurotransmitter and movement is seen with thistechnology. This is shown by using cocaine also in FIGS. 7-11.

Example 6 Serotonin Within Motor Circuits Modulates Rhythmic, EpisodicMovement During Normal Behavior

In FIG. 7, release of 5-HT within DStr, an A₉ DA basal nucleus and nerveterminal field is plotted with ambulations (left panel) and finemovements (right panel). Ambulations are locomotion-movement in thehorizontal plane, around the inside of the behavioral chamber; alsocalled open-field behavior or locomotor activity. These may be monitoredby computerized infrared photocell beams, located around the outside ofthe behavioral chamber. Fine movements are called stereotypic movementsand consist of repetitious movements and/or rhythmic movements, e.g.rearing, chewing, sniffing, and grooming.

These studies were performed in real time during open-field locomotor(exploratory) and stereotypic behaviors from time 0 min to time 60 minas movement occurred. Serotonin release in this motor nucleus isrhythmic with movement even as movement waxed and waned. This was anintriguing and exciting result since locomotion is known to be not onlyrhythmic but, very importantly, it is known to be episodic, unlike mostother rhythmic and repetitive behaviors.

In FIG. 8, release of 5-HT within NAcc core, an A₁₀ DA basal nucleus andnerve terminal field is plotted with ambulations (left panel) and finemovements (right panel). NAcc core is a motor nucleus, as is DStr. Thesestudies, also performed in real time, show that 5-HT was released in amotor nucleus, again, rhythmically and episodically during theopen-field paradigm study of locomotor (exploratory) and stereotypicbehaviors as movement occurred, and again, even as movement's episodicnature was clear. Both FIGS. 7 and 8 show that 5-HT release was rhythmicwith both movement and cessation of movement over the habituation periodthat was initiated and continued from time 60 min to time 120 min.Although the frequency of ambulations and fine movements was notsignificantly different between studies shown in FIGS. 7 and 8, theextent of 5-HT released in A₉ was dramatically less than that releasedin A₁₀ basal nucleus.

The data show a correlation between the 5-HT released within basalnuclei and the motor performance of the animal in the open-fieldparadigm. Moreover, the rhythm between 5-HT released within A₉ basalnucleus and motor behavior is remarkably similar to that rhythm seenbetween 5-HT released within A₁₀ basal nucleus and motor behavior.Similar results were expected from these A₉ and A₁₀ core because theBroderick probe indicator electrode was implanted in NAcc core, themotor-related area of NAcc.

Thus, 5-HT within terminal basal nuclei affects rhythmic movement duringthe normal/natural operation of repetitive motor behaviors. Serotoninrelease within the A₉ terminal field, DStr, and the A₁₀ terminal field,v1NAcc, increased as each open field behavior increased. The 5-HT isreleased within basal nuclei in synchrony with the changes in motorbehavior controlled by the same nuclei.

Lucki, in a 1998 review, states, “diminished 5-HT causes increasedexploratory or locomotor activity” (Lucki I, 1998, Biol. Psychiat.44:151-162). But, there are considerable data that show that 5-HTincreases as exploratory or locomotor movement increases. See e.g.,Bouhuys A L et al., 1977, Physiol. Behav. 19:535-541; Kohler C et al.,1978, Pharmacol Biochem. Behav. 8:223-233; Lorens S A et al., 1976,Brain Res. 108:97-113; Schlosberg A J et al., 1979, J Pharmacol. Exp.Therap. 211:296-304; Srebro B et al., 1975, Brain Res. 89:303-325;Hillegaart V et al., 1989, Pharmacol. Biochem. Behav. 32:797-800;Yeghiayan S K et al., 1997, Pharmacol. Biochem. Behav. 56:251-259. Thetechnology provided here enables scientists to move away from grossbehavioral studies which simply average events over long periods of timeand space.

In FIG. 9, 5-HT release within A₁₀ somatodendrites is plotted withambulations (left panel) and fine movements (right panel). These datashow that 5-HT was released in a motor brain stem nucleus rhythmicallyand episodically during the usual, normal/natural operation of movementbehaviors. Habituation brought about a decrease in 5-HT release as wellas a decrease in locomotor and stereotypic behaviors. Serotonin releasein VTA was less than that seen in the basal nuclei.

Within DA somatodendrites, release of 5-HT dramatically increased in asynchronous and rhythmic manner with ambulations and fine movementbehaviors of grooming and sniffing. Yet, the temporal relationshipbetween 5-HT released within A₁₀ somatodendrites, VTA, with movement isdifferent from that 5-HT released in A₁₀ terminals and in As terminals,with movement. Still highly rhythmic, 5-HT release within A₁₀somatodendrites affects movement in a juxtaposed pattern that was notseen in basal nuclei DA nerve terminals, A₉ DStr or within A₁₀ NAcccore. This was also an intriguing and exciting result since A₁₀somatodendrites are not basal nuclei; A₁₀ cell bodies is a brain stemnucleus, comprised of DA somatodendritic neurons projecting to A₁₀ basalnuclei.

Increased somatodendritic 5-HT cell firing within 5-HT somatodendriteswhich occurs before movement behavior occurs (Jacobs B L et al., 1991,Pharmacol. Rev. 43:563-578) and influences DA interactions withinterminal basal nuclei (Broderick P A et al., 1997, Neuroscience andBiobehavioral Reviews 21(3):227-260) may be an important mechanismmanaging the communication between 5-HT released within DAsomatodendrites during concurrent open-field ambulatory and finemovement behavior. Also, the dendritic release of DA somatodendriticautoreceptors on DA cells (Grace A et al., 1985, NeurotransmitterActions in the Vertebrate Nervous System, Chapter 9) with calciumconductance properties, typical of cells exhibiting dendritic release ofneurotransmitter (Llinas R et al., 1984, Brain Res. 294:127-132), may bean important component of DA neuronal responsiveness. The short time lagcould be due to suppressed DA somatodendritic excitability influenced bydorsal raphe (DR) stimulation (Trent F et al., 1991, Exp. Brain Res.84:620-630).

From FIGS. 7 through 9, a synopsis of important messages aboutnormal/natural 5-HT release within two basal nuclei, a brain stemnucleus and movement behaviors follows.

5-HT released within A₉ and A₁₀ core basal nuclei and A₁₀somatodendrites, increased with locomotor behavior and with the finemovement stereotypic behaviors of grooming and sniffing; 5-HT decreasedduring habituation when movement had essentially ceased.

5-HT release within A₉ and A₁₀ (core) basal nuclei exhibits rhythmicityin synchrony with locomotion and stereotypic behavior; dramaticallysimilar rhythmic patterns occurred within both basal nuclei.

5-HT released within A₁₀ somatodendrites, DA cell bodies, VTA, alsoshowed remarkable rhythmicity with movement and stereotypic behavior,but the rhythmic control by 5-HT in A₁₀ somatodendrites assumes adifferent pattern that that pattern observed when basal nuclei werestudied. VTA is brain stem nucleus and not a basal nucleus.

Data demonstrate normal/natural rhythmic episodic movement behaviors,which previous technologies did not enable.

5HT release within A₁₀ (core) DA nerve terminals during movement andstereotypic behaviors was greater than within A₉ DA nerve terminals.5-HT release within A₉ DA nerve terminals was greater than within A₁₀somatodendrites during movement behaviors.

The data suggest that 5-HT may control episodic and rhythmic movementbehaviors in DA basal nuclei and in the brain stem nucleus, A₁₀somatodendrites. This control or modulation is different in basal nucleicompared with the brain stem nucleus, A₁₀ somatodendrites.

Superior temporal resolution is a crucial component of technologies thatclaim to study neurotransmitters and behavior within the same animal andin real time.

Example 7 Cocaine Disrupts Normal Rhythmic, Episodic Modulation ofMovement via 5-HT in Motor Circuits

Methods used in this example have been described (Broderick P A, 1995,U.S. Pat. No. 5,433,710; Broderick P A, 1996, EP 90914306.7; Broderick PA, 1999, U.S. Pat. No. 5,938,903; Broderick P A, 1989, Brain Res.495:115-12 1; Broderick P A, 1989, Brain Res. 495:115-121; Broderick PA, 1988, Neurosci. Lett. 95:275-280; Broderick P A, 1990,Electroanalysis 2:241-245; Broderick P A, 1993, Pharmacol. Biochem.Behav. 46:973-984; Broderick P A, 2002, Handbook of Neurotoxicology,Vol. 2, Chapter 13; Broderick P A et al., 2000, Brain Res. 878:48-63).The dosage of cocaine used was 10 mg/kg ip.

The temporal synchrony between 5-HT release in basal nucleus A₁₀terminals, v1NAcc, and movement behaviors is disrupted after cocaineadministration. FIG. 10 shows the 5-HT response to cocaine, plotted withresulting ambulations (left panel) and fine movement (right panel)during the psychomotor effects of cocaine, as movement occurred (sameanimal control in real time). Comparing this figure with FIG. 8 revealsthat normal communication between basal 5-HT release in NAcc andmovement behaviors is disrupted by cocaine. Although 5-HT levels stillincrease after cocaine, rhythmic control of movement by 5-HT is nolonger observable in either ambulatory (locomotor) or fine movement(stereotypic) behaviors. Moreover, the magnitude of the 5-HT increaseafter cocaine is significantly less than 5-HT released and observedduring natural movement without cocaine. Cocaine-induced behaviors afterhabituation are still increased, but the behaviors seem to occur in afrequency similar to those frequencies usually observed in smaller,younger, animals.

The temporal synchrony between 5-HT release in A₁₀ somatodendrites andmovement behaviors is also disrupted after cocaine administration.Although VTA is not a basal nucleus, these DA cell bodies are acomponent of the mesocorticolimbic motor circuit. FIG. 11 shows the 5-HTresponse to cocaine, plotted with resulting ambulations (left panel) andfine movement (right panel) behaviors during the psychomotor effects ofcocaine, as movement occurred (same animal control in real time). Thisfigure shows that the previous normal/natural communication (shown inFIG. 9) between basal 5-HT release in A₁₀ somatodendrites and movementbehaviors has been disrupted. The data show that enhanced 5-HT releasein VTA after cocaine is no longer synchronous with movement behaviors.Thus, ambulations (locomotion) and fine movement behaviors of groomingand sniffing are not related temporally to 5-HT release at A₁₀somatodendrites after cocaine. Serotonin release increased when comparedwith habituation behavior, but the magnitude of the increase was smallerthan observed during normal movement without cocaine. Despite the risein 5-HT levels, 5-HT does not direct normal, rhythmic episodic movementsafter cocaine administration.

The general trend of changes in 5-HT and movement behaviors observedhere confirm previously reported results (Yeghiayan S K et al., 1997,Pharmacol. Biochem. Behav. 56:251-259). However, previous studies havenot been able to detect these subtle changes nor have these previousstudies been able to detect normal/natural episodic, rhythmic nature oflocomotor (exploratory) movement or stereotypy, either in neurochemistryor behavior.

5-HT control or modulation of movement behaviors in A₁₀ basal nucleusand in A₁₀ somatodendrites during normal/natural movement behaviors issubsequently disrupted by cocaine. Even in the first thirty minutesafter cocaine, the episodic rhythmic nature of locomotor (exploratory)movement behavior and stereotypic behavior has been disrupted.

5-HT release in basal nuclei and VTA DA somatodendrites after cocaine isgreater than those during habituation but less than those seen duringnormal/natural movement behaviors.

Neuroadaptation cannot be determined by simply studying the generaldirection of the response of 5-HT to cocaine. Using Broderick probemicrovoltammetry, neuroadaptative responses by 5-HT in motor circuitshave been seen after a single injection of cocaine. The observedneuroadaptative response by 5-HT in motor circuits is independent oftemperature changes since temperature was kept constant at 37.5±0.5° C.Neuroadaptation may be a predisposition to cocaine neurotoxicity.

In vivo microvoltammetric studies enable the detection of subtle changesnecessary to see alterations in normal/natural neurochemistry andbehavior that existed before the administration of cocaine. The studiesshow that neuronal damage to basal nuclei and brain stem nuclei may haveoccurred after the administration of cocaine.

Superior temporal resolution is a crucial component of technologies thatclaim to study neurotransmitters and behavior within the same animal andin real time.

Example 8 CPGs Thin Basal Nuclei May Induce Rhythmic Movement by 5-HT

The following provides a rationale for studies using CPGs to explain howbrain and spinal cord injuries can come about. The following alsodescribes the necessity for neurotransmitters and movement, behavior orotherwise to be synchronous. The invention further provides methods fordiagnosing injury where the patterns are asynchronous.

To date, there have been no reports of 5-HT modulation that is intrinsicto CPG's that operate rhythmic locomotion or stereotypy in basal nuclei.Yet, basal nuclei are known to be involved in the development ofautomaticity and to play a primary role in both movement preparation andexecution, possibly by optimizing muscular activity patterns once amotor decision has been made (Brooks D J, 1996, Basal Ganglia functionduring normal and Parkinsonian movement, PET activation studies. InAdvances in Neurology (Battistin, L. Scarlato, G., Caraceni, T. andRuggieri, S. Eds.), Lippincott-Raven, Phil., Pa. pp. 433-441).

Studies of neurotransmitters in basal nuclei and brain stem nuclei havetypically focused on DA. For example, it is known that the basic rhythmfor locomotion is generated centrally in spinal networks. The transitionfrom stance to swing is regulated by afferent signals from leg flexorand extensor muscles. These afferent signals are ultimately influencedin intensity and pattern by descending signals from CNS neuronalcircuitry (Pearson K et al., 2000, Principles of Neural Science, 4thedition, pp. 738-755). Again, the catecholamines have taken preferenceas targets for study. Landmark studies, performed about thirty yearsago, showed that injection of the catecholaminergic drugs, L-DOPA andnialamide, into spinal cord generated spontaneous locomotor activity(Jankowska E et al., 1967, Acta Physiol. Scand. 70:369-388; Jankowska Eet al., 1967, Acta Physiol. Scand. 70:389-402).

Moreover, electrophysiological studies of DA in the basal nucleus, DStr,have shown that DA neurons operate in bursts of action potentials thatincrease bursting and change bursting patterns when 96% DA neurons aredamaged (Hollermann J R et al., 1990, Brain Res. 533:203-212). Otherelectrophysiological studies have shown that the excitotoxin, kainicacid, when injected into the basal nucleus, DStr, changed the pattern ofthe normal neuronal rhythm in the basal stem nucleus, substantia nigra(SN). Since SN usually exhibits a slow rhythmic firing of actionpotentials, damage to the neurons has been reported to cause adisorganized rhythm. This SN model has been used as an animal model tostudy the movement disorder, Huntington's Disease (Doudet D et al.,1984, Brain Res. 302:45-55). Therefore, empirical precedents provideevidence for a clear association between DA, neuronal damage, anddisorganized rhythms, at least from electrophysiological studies.

The instant invention provides a novel approach of examining theinvolvement of 5-HT modulation in the functioning of CPGs that operaterhythmic locomotion or stereotypy in basal nuclei. The technologypresented here, provides a way to concurrently study 5-HT release inbasal nuclei, movement circuits, and behavior, in an animal or human.The analysis may further include observing changes in one or more ofthese three factors upon exposure to some stimulus.

The empirical evidence presented in the Examples suggests that (a) 5-HTin basal nuclei may be responsible, at least in part, for thenormal/natural episodic, rhythmicity known to exist with locomotor andstereotypic movements and (b) a subtle neuroadaptation is caused bycocaine between 5-HT and cocaine-induced movements known as “psychoticbehavior” by a single or multiple CPG network. Interestingly,neuroadaptation, induced by cocaine is highly time-dependent. This isconsistent with the observation that neuromodulatory inputs canreconfigure CPG networks to produce specific motor output patterns(Kiehn O et al., 1996, J. Neurophysiol. 75:1472-1482).

Thus, cocaine may act through a time-dependent, 5-HT CPG neuronalnetwork to cause neuroadaptation to occur which may lead to cocaineneurotoxicity. This cocaine-5-HT driven neuroadaptation may reflectneuronal damage and may be a marker for cocaine neurotoxicity.

Locomotor (exploratory) activity and stereotypic behaviors are episodicand rhythmic although they are commonly not described as such. FIGS. 7through 9 depict the episodic, rhythmicity seen in normal/naturalmovement behaviors. Neuromodulation by the biogenic amine, 5-HT, withinbasal nuclei and A₁₀ brain stem nucleus is depicted as these movementsoccur and as these movements are presumably controlled, directed,modulated or regulated by 5-HT.

Hyperactive locomotion and stereotypic behaviors are often thought ofand referred to as psychomotor stimulant behaviors. Also, repetitivebehaviors induced by cocaine have been perceived as “meaningless” orgoalless behaviors. It has been said that the term “stereotypy” appliesto a behavioral act that is repeated again and again, but, unlike amotivational act, it makes no sense because it does not achieve anadaptive outcome (Teitelbaum P, Pellis S M, and De Vietti T L, 1990,Disintegration into sterotypy induced by drugs of brain damage: amicrodescriptive behavioral analysis. In: Neurobiology of SterotypedBehavior (Cooper S J and Dourish C T Eds.), Oxford Univ. Press, NY. pp.169-199.). However, repetitive behaviors produced by cocaine may not bemeaningless or non-adaptive. Rather, neuroadaptation to cocaine,possibly leading to habit-forming behavior, may be the unfortunatemaladaptive outcome. FIGS. 10 and 11 show that cocaine disrupted thenormal episodic rhythm of natural movement; cocaine caused normalrhythmic movement to be disorganized.

It is interesting that the concept of “rhythm” in normal open-fieldmovement is virtually ignored or forgotten when one studies theliterature on the mechanism of action of cocaine. Conceptually, whenneuroscientists speak of cocaine-induced psychomotor stimulant behavior,it seems as if movement does not occur until cocaine is administered orinjected. The Examples herein illustrate that there are actually greaterenhancements in 5-HT during movement before cocaine administration thanin the same animal after cocaine administration. In addition,cocaine-induced movement was not significantly increased over movementwithout cocaine.

Example 9 Schizophrenia

The first evidence that schizophrenia may be associated with 5-HTergicabnormalities was the observation that there was a structural similaritybetween 5-HT and the hallucinogenic drug, lysergic acid diethylamide(LSD) (Gaddum J H, 1954, Ciba Foundation Symposium on Hypertension, pp.75-77; Wooley D W et al., 1954, PNAS 40: 228-231). Solomon Snyder(Snyder S H, 1972, Arch. Gen. Psychiat. 45:789-796) reported, however,that the psychosis induced by LSD in humans exhibited a vastly differentsymptomatology that than of schizophrenic-induced psychosis. A revivalof interest in the relationship between 5-HT and schizophrenia occurredabout fifteen years ago, when the atypical neuroleptic was found to havea high affinity for 5-HT₂ receptors (Altar C A et al., 1986, Brain Res.Bull. 16:517-525). Also, clozapine was found to be particularlyeffective in treating patients intractable to other neuroleptics andclozapine was found to produce less extrapyramidal side effects (EPS)(movement disorders) than did other previous neuroleptics (Kane J etal., 1988, Arch. Gen. Psychiat. 45:789-796; Tamminga C A et al., 1987,Psychopharmacology: the Third Generation of Progress, pp. 1129-1140).

A mediation for 5-HT in either the disease of schizophrenia itself or inthe movement disorders known to be caused by the classical neurolepticsremains under study; two excellent reviews are published (Iqbal N etal., 1995, Eur. Neuropsychopharmacol. 5(Suppl.):11-23; Abi D A et al.,1977, J. Neuropsychiat. Clin. Neurosci. 9:1-17). Nonetheless, a currenthypothesis derived from human and animal studies regarding this atypicalneuroleptic is that clozapine acts via its 5-HT₂ antagonistic effect toalleviate movement disorders in psychosis. Furthermore, in treating theschizophrenic psychotic abnormality via its DAD₂/5-HT₂ antagonisticreceptor action, the drug produces less EPS than its classicalcounterparts. (Meltzer H Y, 1989, Psychopharmacology 99(Suppl.):18-27;Broderick P A et al., 1998, J. Neural. Transm. 105:749-767; Hope O.,Lineen, E., Okonji, C., Green, S., Saleem, A., Aulakh, C. S. andBroderick, P. A., 1995, Cocaine has remarkable nucleus accumbens effectson line, with behavior in the serotonin-deficient Fawn Hooded rat.NIH/NIGMS Symposium, Washington, D.C; Wadenburg M L, 1996, Neurosci.Biobehav. Rev. 20:325-329; Kapur S et al., 1996, Am. J. Psychiat.153(4):466-476; Martin P, 1998, 5-HT₂ Receptor Antagonism andAntipsychotic Drugs; A Behavioral and Neurochemical Study in a RodentHypoglutamatergia Model. PhD. Thesis, Goteborg Univ., Sweden, pp. 1-64)and possibly by its DAD₄ action (Van Tol HHM et al., 1991, Nature350:610-614). Perhaps the present data can lend an explanatory note to afairly recent study in which the classical DAD₂ receptor antagonist,haloperidol, was shown to induce the movement dysfunction, catalepsy, by5-HTergic mediation (Neal-Beliveau B S et al., 1993, J. Pharmacol. Exp.Therap. 265:207-217).

Example 10 Conclusion

Serotonin release from DA A₉ and A₁₀ basal nuclei and A₁₀ brain stemnuclei may be regulated by a 5-HT-regulated CPG, presumably originatingfrom raphe somatodendrites based on empirical studies using in vivomicrovoltammetry with Broderick probe electrodes. Animals exhibitedrepetitive, episodic and rhythmic normal/natural movements, influencedby 5-HT within DA A₉ and A₁₀ neural circuits without any drug treatment.Furthermore, cocaine disrupted such normal/natural episodic rhythmicmovement by altering release of 5-HT, precisely within the DA basalnuclei that are responsible for controlling voluntary movement. Afurther disruption of normal, episodic rhythmic movement by 5-HT aftercocaine occurred in the brain stem nucleus, VTA, the cell bodies for thebasal nucleus, NAcc. Thus, 5-HT neuroadaptation by cocaine may be apredisposition or marker for cocaine induced neuronal damage orneurotoxicity. Implications for the study of other movement disorders,like spinal cord injury, through these empirical data, are noteworthy.

Example 11

In an empirical protocol of EEG and functional mapping, all subjectsevaluated to have medically intractable partial seizures will have scalpEEG electrodes or BRODERICK PROBE® sensors applied according to the10-20 system with the addition of inferior temporal and/or sphenoidalelectrodes.

Digital EEGs will be recorded and analyzed with a 64 channel TelefactorBeehive and Beekeeper system (Telefactor Corporation, PA) with a 200 Hzsampling rate and stored on VHS tape.

Intracranial EEGs will be simultaneously recorded, under anesthesia,with a modified 32 channel, 400 Hz sampling rate Telefactor unit inorder to record gamma frequency activity.

Subjects having placement of subdural strip, grid or depth electrodesunder anesthesia will have scalp and sphenoidal electrodessimultaneously placed.

Scalp and intracranial EEG recordings will be analyzed using aTelefactor Beekeeper System in both bipolar and referential montages.

Simultaneous intracranial, sphenoidal and scalp EEGs will be reviewed tostudy the ictal, scalp EEG manifestations of seizure onsets from variousneocortical regions. The specificity of inferior temporal and sphenoidalelectrodes for predicting mesial temporal versus temporal neocortical ororbitofrontal seizure onsets will also be examined.

Language, motor and sensory functions will be mapped when clinicallyindicated with electrical stimulation using a Grass Instrument S-12stimulator (Astromed (Grass-Telefactor); West Warwick, R.I.).

Intracranial electrodes and scalp electrode locations will be referencedto the MRI and thereby to each other.

Intraoperative Localization utilizes the same subjects. In the operatingroom, data gathered during the pre-surgical evaluation will be availableto the surgeon.

The anesthesiologist will appropriately anesthetize the patient (generalinhalational anesthetic or perhaps the intravenous sedative, propofol)and after the appropriate length of time to allow the pharmacokineticinduction of the anesthetic, the surgeon will use the Polhemuslocalization device and the same fiducials used to co-register theelectrophysiological and anatomic data.

The skull of the patient located behind the ear, encases the neocortex.The skull will be removed by the surgeon and placed in a −80 degreeCentigrade freezer.

Now, the surgeon may place the electromagnetic sensor of thelocalization device at any point on the brain and automatically displaythe corresponding MRI image on the desk-top computer with or without thesuperimposed pre-surgical findings.

Although any region of the brain may be imaged and/or recorded, theneocortex will specifically examined. The neocortex is divided into sixlayers. In this study, the gray matter in the outer layers of theneocortex is the basis of this example. White matter will be avoidedwith the use of the BRODERICK PROBE® sensors which can differentiate,within seconds, between gray and white matter according to differentialwaveforms produced in gray versus white matter.

With the neocortex now clearly visible, the instant inventive BRODERICKPROBE® sensor will be carefully inserted approximately 5 or 6 mm intothe outer layers, preferably layer number one.

Specifically, the inventive sensor comprises graphite, nujol oil(containing tocopherol, vitamin E), and the 12 carbon chain, unsaturatedsurfactant fatty acid, lauric acid, both with and without pretreatmentof the lipid surfactant, phosphotidylethanolamine.

A (micro) reference electrode will be placed approximately 6-7 mm fromthe instant inventive sensor and a stainless steel auxiliary will beplaced in an adjacent part of the cortex, about 6-7 mm from theindicator sensor, but simply only about 2 mm deep. The positioning ofthe three electrode assembly will be triangular (see, FIG. 12).

Neuromolecular images, based on electrochemistry of electron-transferwill take place every 3 minutes for a period of 20 to 30 minutes. Eachimage will take about 40-60 seconds. The scan rate will be 10 millivoltsper second.

A semiderivative circuit, mathematically derived from the linear sweepcircuit (Autolab, Brinkmann, Long Island) will be used to apply voltagesin millivolts to activate the instant inventive indicator sensor.

Charging current will be eliminated in the first 20 seconds in negativepotentials.

The silver/silver chloride (micro) reference will provide the relativezero point to estimate changes in current produced by the indicatorsensor of the instant invention.

Stainless steel auxiliary electrode will provide an electrical ground.

Neuromolecular images, electrochemical signals will be selectivelydetected at specific millivoltage potentials, dependent on specificoxidation properties of biomolecules.

Images will be automated, remotely recorded and integrated statisticallywith the Autolab system (Brinkmann; Long Island, N.Y.).

Subtypes of epilepsy and tumors will be defined on the basis of theneurochemical data combined with the electrophysiological data.

The cortical resection will then be tailored with the benefit ofcontinuous visualization of the underlying anatomy.

The cortical resection will then be tailored as well, with the benefitof the instant inventive steps of the sensors, i.e., epileptogenic zoneswill be more clearly delineated with neurochemical data combined withvisual inspection by the surgeon.

Surgical procedures will continue to completion and the patient will bereturned to the epilepsy unit wherein the patient will recover fromanesthesia.

Therefore, the BRODERICK PROBE® sensors will assist image-guidedneurosurgery and perform functionally in a novel and unique way in theoperating room. These sensors are less cumbersome and less expensivethan is MRI. Nonetheless, it is important to note that NeuromolecularImaging) (NMI) with said sensors, is similar in accurate functionalityto MRI in that NMI uses electron transfer and MRI uses proton transfer.(Broderick, P. A., Pacia, S. V., “Imaging white matter signals inepilepsy patients: A unique sensor technology”. In: Broderick, P. A.,Rahni, D. N. and Kolodny, E. H. (Eds.) Bioimaging in Neurodegeneration.Humana Press Inc. Totowa, N.J. (2005)).

The above description of various embodiments has been presented forpurposes of illustration and description. It is not intended to beexhaustive or limiting to the precise forms disclosed. Obviousmodifications or variations are possible in light of the aboveteachings. The embodiments discussed were chosen and described toprovide illustrations and its practical application to thereby enableone of ordinary skill in the art to utilize the various embodiments andwith various modifications as are suited to the particular usecontemplated. All such modifications and variations are within thesystem as determined by the appended claims when interpreted inaccordance with the breadth to which they are fairly, legally andequitably entitled.

1. A sensor comprising: a) a construction; and b) an indicator.
 2. Thesensor of claim 1, wherein the sensor is partially or fully encased inan encasement, and the encasement is a hollow three-dimensional surface.3. The sensor of claim 2, wherein the hollow three-dimensional surfacehas an end face in any geometric shape.
 4. The sensor of claim 3,wherein the geometric shape comprises: a circle, a triangle, aquadrilateral, a rhombus, a parallelogram, a rectangle, or a polygon. 5.The sensor of claim 2, wherein the encasement comprises conductingmaterial, semi-conducting material, non-reactive material, ornon-reactive polymers, or combinations thereof.
 6. The sensor of claim5, wherein the encasement comprises: metals, polymers, or blendsthereof, polytetrafluoroethylene, fluorinated ethylene-propylene,perfluoroalkoxy polymer resin, polymethylmethacrylate,polyethylethacrylate, steel, stainless steel, silicon, germanium,silver, platinum, gold, or combinations thereof.
 7. The sensor of claim1, wherein the construction comprises: a conducting material,semi-conducting material, conducting metal, or a semi-conducting metal,or combinations thereof.
 8. The sensor of claim 7, wherein theconstruction comprises: steel, stainless steel, silicon, germanium,silver, platinum, or gold, or combinations thereof.
 9. The sensor ofclaim 1, wherein the indicator comprises: a) at least one form ofcarbon, or combinations thereof; and b) at least one lipid or an entityhaving a lipid, or combinations thereof.
 10. The sensor of claim 9,wherein the carbon comprises: graphite, fullerenes, cylindricalfullerenes, buckminsterfullerenes, buckyballs, nanotubes, probingtubes,cold form carbon steel, white carbons, dioxosilane, or diamonds, orcombinations thereof.
 11. The sensor of claim 9, wherein the lipidcomprises: lipids, fats, oils, animal fats or oils, plant fats or oils,mineral oils, glycerol containing lipids, membrane lipids, soaps ordetergents, waxes, cells, cell components, stem cells, electroplaques,lipoproteins; or combinations thereof.
 12. The sensor of claim 11,wherein the lipid comprises: lipids, entities having lipids, fats, oils,animal fats or oils, plant fats or oils, mineral oils, nujol oil,glycerol containing lipids, membrane lipids, soaps or detergents, waxes,cells, cell components, stem cells, electroplaques, lipoproteins, fattyacids, glycerides, monoglycerides, diglycerides, triglycerides,artificial or synthesized fats or oils, heifer fats, ox-depot fats,Valeria indica fats, tallow, red tallow, Malabar tallow, vegetabletallow, cocoa butter, soybean oil, safflower oil, sesame oil, peanutoil, coconut oil, linoleic acid, linoleic acid in vegetable oil, soybeanoil, cottonseed oil, corn oil, or poppyseed oil, lauric acid, lauricacid in coconut oil, cholesterol, phosphotidylcholine,phosphotidylethanolamine, sphingomyelin, lecithin, lysolecithin,steroids, isoprenoids, eicosenoids, sodium alkyl benzene sulfonate,sodium lauryl sulfate, jejoba wax comprised of gadoleic acid,N-stearoylcerebroside, N-stearoylsphingosine, cardiolipin, orcombinations thereof.
 13. The sensor of claim 1, wherein the indicatoris pre-treated, inserted, or coated with at least one biomolecule, orcombinations thereof.
 14. The sensor of claim 13, wherein thebiomolecule comprises: a pharmaceutical compound, neurotransmitters,neuromodulators, nucleic acids, hormones, vitamins, surfactants, soaps,detergents, stabilizing proteins, amyloid proteins, or combinationsthereof.
 15. The sensor of claim 14, wherein the biomolecule comprises:pharmaceutical compounds, pharmaceutical compounds specific forneurodegenerative or neuropsychiatric diseases, disorders, andconditions, neurotransmitters, neuromodulators, hormones, surfactants,soaps, detergents, pramipexole, topiramate, clozapine, dopamine,serotonin, norepinephrine, acetylcholine, adenosine, estrogen, vitamins,vitamin A, vitamin E, brain lipids, phosphotidylethanolamine, tallow,sodium lauryl sulfate, N-acetyl-aspartate, choline, lactate, uric acid,stabilizing proteins, amyloid proteins, ascorbic acid, γ-aminobutyricacid, glutamate, neurotensin, somatostatin, dynorphin, homovanillicacid, nucleic acids, tryptophan, tyrosine, nitrous oxide, nitric oxide,or combinations thereof.
 16. A method for microvoltammetric imaging ofchanges in biomolecule concentrations in response to diagnosticchallenge or therapeutic treatment comprising: exposing a neural cell,body, blood, or urine to a diagnostic challenge or therapeutictreatment; contacting said cell, body, blood, or urine with a sensor ofclaim 1; applying a potential to the sensor; and monitoring a temporallyresolved microvoltammogram.
 17. A diagnostic method for monitoringneural functions in a mammal comprising: contacting neural cell, body,blood, or urine of said mammal with a sensor of claim 1; applying apotential to said sensor; and generating a temporally resolvedmicrovoltammogram, wherein the microvoltammogram indicates the status ofneural function in the mammal.
 18. A method of diagnosing and/ormonitoring a neurological disease, disorder, or condition, comprising:generating a temporally resolved microvoltammogram of a subject;determining from said microvoltammogram the presence and concentrationof at least one biomolecule; and comparing said biomoleculeconcentration(s) to specific threshold values of each biomolecule orbiomolecules to determine the presence of statistically significantconcentration differences, wherein said threshold values are derivedfrom the sensor microvoltammogram(s) of at least one healthy individual,wherein the microvolatammogram uses the sensor of claim
 1. 19. Themethod of claim 18, wherein the number of biomolecules is at least two.20. The method of claim 18, wherein the biomolecule comprises:pharmaceutical compounds, pharmaceutical compounds specific forneurodegenerative or neuropsychiatric diseases, disorders, andconditions, neurotransmitters, neuromodulators, hormones, surfactants,soaps, detergents, pramipexole, topiramate, clozapine, dopamine,serotonin, norepinephrine, acetylcholine, adenosine, estrogen, vitamins,vitamin A, vitamin E, brain lipids, phosphotidylethanolamine, tallow,sodium lauryl sulfate, N-acetyl-aspartate, choline, lactate, uric acid,stabilizing proteins, amyloid proteins, ascorbic acid, γ-aminobutyricacid, glutamate, neurotensin, somatostatin, dynorphin, homovanillicacid, nucleic acids, tryptophan, tyrosine, nitrous oxide, nitric oxide,surfactants, or combinations thereof.
 21. The method of claim 18,wherein the biomolecule detects conditions, diseases, or disorders of:the basal ganglia, athetoid, dystonic diseases, Parkinson's disease,Huntington's disease, epilepsy, Lesch-Nyhan disease,controlled-substance addictions, cerebral ischemia, white matterdisease, stroke cerebral hemorrhage, head trauma, multiple sclerosis,central nervous system infection, hydrocephalus, Leukodystrophies, andneoplasms.
 22. The method of claim 21 wherein the controlled substanceaddiction is an addiction to a controlled substance comprising opiates,stimulants, or depressants.
 23. A method for detecting a site of nerveor neuronal damage or blockage in a mammal having or being at risk ofdeveloping nerve damage or blockage comprising: generating a temporallyresolved microvoltammogram of a tissue of said mammal; simultaneouslymonitoring movement behavior of said mammal; and comparing saidmicrovoltammogram and movement behavior to a reference microvoltammogramof corresponding tissue of a healthy individual and concurrent referencemovement behavior of said healthy individual, wherein themicrovoltammogram uses the sensor of claim
 1. 24. The method of claim 23wherein the nerve damage or blockage is a physical injury or blockage.25. The method of claim 24 wherein the physical injury or blockage is aspinal cord injury or blockage.
 26. The method of claim 24 wherein thenerve damage or blockage is a chemically-induced injury or blockage. 27.A diagnostic method for brain or spinal cord injury comprising:generating a temporally resolved microvoltammogram of a tissue of amammal having or being at risk of developing a brain or spinal cordinjury; simultaneously monitoring movement behavior of said mammal; andcomparing said microvoltammogram and movement behavior to a referencemicrovoltammogram of corresponding tissue of a healthy individual andconcurrent reference movement behavior of said healthy individual,wherein the microvoltammogram uses the sensor of claim
 1. 28. The methodof claim 27 wherein the movement behavior is ambulation, fine motormovement, or combinations thereof.
 29. A diagnostic method for braincancer comprising: generating a temporally resolved microvoltammogram ofcancerous brain cells or tissue; determining from said voltammogram thepresence and concentration of at least two biomolecules, wherein saidbiomolecule comprises: pharmaceutical compounds, neurotransmitters,neuromodulators, hormones, surfactants, soaps, detergents, dopamine,serotonin, norepinephrine, acetylcholine, adenosine, estrogen, vitamins,vitamin A, vitamin E, brain lipids, phosphotidylethanolamine, tallow,sodium lauryl sulfate, N-acetyl-aspartate, choline, lactate, uric acid,stabilizing proteins, amyloid proteins, ascorbic acid, γ-aminobutyricacid, glutamate, neurotensin, somatostatin, dynorphin, homovanillicacid, nucleic acids, tryptophan, tyrosine, nitrous oxide, nitric oxide,or combinations thereof, and comparing said biomolecule concentrationsto specific threshold values of each of the biomolecules to determinethe presence of statistically significant concentration differences,wherein said threshold values are derived from the microvoltammogram(s)of healthy cells or tissue and said step of comparing said biomoleculesdistinguishes whether the cancerous cells are present in gray matter orwhite matter, wherein the microvolatammogram uses the sensor of claim 1.30. The method of claim 29, wherein the brain cancer comprises:malignant gliomas, astrocytomas, oligodendogliomas, ependymomas,gliosarcoma, meningioma, hammartomas, ganglioglioneurocytomas, primitiveneuroectodermal tumors (pnet), medulloblastomas, neurofibromas,schwannomas, neuromas, teratomas, pituitary adenomas, or metastatictumors.
 31. The method of claim 29 wherein said biomolecules are atleast norepinephrine and dopamine.
 32. The method of claim 29 wherein atleast one of said biomolecules is serotonin.
 33. The method of claim 29wherein said markers are at least norepinephrine and serotonin and saidcomparing indicates gray matter if the catecholamine peak is about halfthe amplitude of the catecholamine reference peak of white matter andthe serotonin peak is about double the amplitude of the serotininreference peak of white matter.
 34. A method of measuring theneurotoxicity of a substance comprising: comparing a temporally resolvedmicrovoltammogram of neural tissue in the absence of said material witha temporally resolved microvoltammogram of tissue in the presence ofsaid material, wherein the microvolatammogram uses the sensor ofclaim
 1. 35. A method of diagnosing epilepsy comprising: generating atemporally resolved microvoltammogram of a tissue of a subject; andcomparing said microvoltammogram to at least one referencemicrovoltammogram; wherein said reference microvoltammogram is of thecorresponding tissue of an individual, comprising: a healthy individual,an individual having mesial temporal lobe epilepsy, an individual havingneocortical temporal lobe epilepsy, an individual having parietal lobeepilepsy, an individual having frontal lobe epilepsy, an individualhaving jacksonian epilepsy, an individual having Rasmussen's epilepsy,an individual having Lafora's body disease, an individual havingLennox-Gestaut, an individual having Landau-Kleffner syndrome, anindividual having West Syndrome, an individual having primarygeneralized epilepsies, an individual having partial epilepsy, or anindividual having post-traumatic epilepsy, wherein the microvoltammogramuses the sensor of claim
 1. 36. The method of claim 35, wherein saidmicrovoltammogram of a subject is compared with more than one referencemicrovoltammogram.
 37. The method of claim 35, wherein saidmicrovoltammogram of a subject is compared with the referencemicrovoltammogram of a healthy individual, an individual having mesialtemporal lobe epilepsy, and an individual having neocortical temporallobe epilepsy.
 38. A diagnostic method for temporal lobe epilepsycomprising: generating a temporally resolved microvoltammogram oftemporal lobe test tissue; determining from said microvoltammogram thepresence and concentration of at least two biomolecules comprising:pharmaceutical compounds, pharmaceutical compounds specific forneurodegenerative or neuropsychiatric diseases, disorders, andconditions, neurotransmitters, neuromodulators, hormones, surfactants,soaps, detergents, pramipexole, topiramate, clozapine, dopamine,serotonin, norepinephrine, acetylcholine, adenosine, estrogen, vitamins,vitamin A, vitamin E, brain lipids, phosphotidylethanolamine, tallow,sodium lauryl sulfate, N-acetyl-aspartate, choline, lactate, uric acid,stabilizing proteins, amyloid proteins, ascorbic acid, γ-aminobutyricacid, glutamate, neurotensin, somatostatin, dynorphin, homovanillicacid, nucleic acids, tryptophan, tyrosine, nitrous oxide, nitric oxide,or combinations thereof; and comparing said test tissue biomoleculeconcentrations to specific threshold values of each of the biomoleculesto determine the presence of statistically significant concentrationdifferences, wherein said threshold values are derived from themicrovoltammogram(s) of tissue selected from the group consisting ofhealthy temporal lobe tissue, mesial temporal lobe epileptic tissue, andneocortex temporal lobe epileptic tissue, wherein the microvolatammogramuses the sensor of claim
 1. 39. The method of claim 38, wherein saidstep of comparing said biomolecule distinguishes whether the test tissueis healthy tissue, mesial temporal lobe epileptic tissue, or neocortextemporal lobe epileptic tissue.
 40. A method for determining theconcentration of a therapeutic material in a brain tumor comprising:contacting said tumor with the sensor of claim 1; applying a potentialto said sensor; generating a temporally resolved sensormicrovoltammogram; and determining from said microvoltammogram theconcentration of said material.
 41. The method of claim 40, wherein saiddetermining comprises calculating the concentration of said materialusing the Cottrell equation.
 42. A method of guiding neurosurgerycomprising: distinguishing gray matter, white matter, tumor tissue,necrotic tissue, ischemic tissue, and edematous tissue, wherein saiddistinguishing comprises: a) generating a temporally resolvedmicrovoltammogram of a test tissue using the sensor of claim 1, an EEGsensor, or combinations thereof; b) determining from saidmicrovoltammogram the presence and concentration of at least twobiomolecules comprising pharmaceutical compounds, neurotransmitters,neuromodulators, hormones, surfactants, soaps, detergents, dopamine,serotonin, norepinephrine, acetylcholine, adenosine, estrogen, vitamins,vitamin A, vitamin E, brain lipids, phosphotidylethanolamine, tallow,sodium lauryl sulfate, N-acetyl-aspartate, choline, lactate, uric acid,stabilizing proteins, amyloid proteins, ascorbic acid, γ-aminobutyricacid, glutamate, neurotensin, somatostatin, dynorphin, homovanillicacid, nucleic acids, tryptophan, tyrosine, nitrous oxide, nitric oxide,or combinations thereof; and c) comparing said test biomoleculeconcentrations to specific threshold values of each of the biomoleculesto determine the presence of statistically significant concentrationdifferences, wherein said threshold values are derived from the sensormicrovoltammogram(s) of reference tissue of gray matter, white matter,tumor tissue, necrotic tissue, ischemic tissue, edematous tissue, orcombinations thereof, wherein the microvolatammogram uses the sensor ofclaim
 1. 43. The method of claim 42, wherein said referencemicrovoltammogram is from a human or non-human mammal having mesialtemporal lobe epilepsy, neocortical temporal lobe epilepsy, parietallobe epilepsy, frontal lobe epilepsy, Rasmussen's epilepsy, Lafora'sbody disease, Lennox-Gestaut, Landau-Kleffner syndrome, west Syndrome,primary generalized epilepsy, partial epilepsy, or post-traumaticepilepsy.
 44. The method of claim 42, wherein said intraoperativeneuroanatomic localization comprises delineating marginal boundaries inbrain substrates.
 45. The method of claim 42 wherein said contactingstep is minimally invasive and occurs through a burr hole, smallcraniotomy, or an incision.
 46. A method for continuous or intermittentmonitoring and administration of pharmacological and nonpharmacologicaltreatments for a neurological disorder in a mammal, comprising: a)contacting neural cells with the sensor of claim 1; b) applying apotential to the sensor; c) generating a temporally and/or spatiallyresolved sensor microvoltammogram; d) interpreting the microvoltammogramwith a microprocessor or computer, wherein said microprocessor orcomputer determines the presence and concentration of a biomoleculecomprising: pharmaceutical compounds, pharmaceutical compounds specificfor neurodegenerative or neuropsychiatric diseases, disorders, andconditions, neurotransmitters, neuromodulators, hormones, surfactants,soaps, detergents, pramipexole, topiramate, clozapine, dopamine,serotonin, norepinephrine, acetylcholine, adenosine, estrogen, vitamins,vitamin A, vitamin E, brain lipids, phosphotidylethanolamine, tallow,sodium lauryl sulfate, N-acetyl-aspartate, choline, lactate, uric acid,stabilizing proteins, amyloid proteins, ascorbic acid, γ-aminobutyricacid, glutamate, neurotensin, somatostatin, dynorphin, homovanillicacid, nucleic acids, tryptophan, tyrosine, nitrous oxide, nitric oxide,or combinations thereof, and compares said biomolecule concentration toa threshold value of said respective biomolecule, and wherein saidmicroprocessor or computer generates an output which administers thepharmacological or nonpharmacological treatment in an amount sufficientto treat said neurological disorder.